Telomelysin/gfp-expressing Recombinant Virus

Abstract

The present invention relates to providing a reagent for cancer cell detection or cancer diagnosis, a pharmaceutical composition for the treatment of cancer, and a method of treating or preventing cancer in a subject. Specifically, the present invention relates to a reagent for cancer cell detection, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome.

Description

[0001] This application claims the benefit of the filing date of JP application 2004-285383 filed on Sep. 29, 2004. JP application 2004-285383 is herein incorporated by reference in its entirety. This application is also a continuation-in-part application of U.S. application Ser. No. 11/158,479 filed on Jun. 21, 2005, incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to a reagent for detecting cancer cells or diagnosing cancers, and a cell death-inducing agent. Also provided is a method of treating or preventing cancer in a subject.

[0003] Telomerase activity is often enhanced in malignantly transformed cells or immortalized cell strains, whereas telomerase activity is hardly detected in normal somatic cells excluding such as germ line cells, blood lineage cells and epithelial cells. Therefore, attempts to detect cancer using telomerase activity as an indicator have been made (Shay J W, Zou Y, Hiyama E, Wright W E. Telomerase and Cancer. Hum Mol Genet. 10 (7): 677-85, 2001).

[0004] On the other hand, detection of cancer tissues and metastatic lymph nodes in vivo has been studied eagerly in the field of diagnostic imaging. For example, biological diagnosis with PET and image analysis fully utilizing neural network have been reported. Further, investigations into the anti-tumor activity and safety of replication-selective viruses have been reported (DeWeese T L, van der Poel H, Li S, Mikhak B, Drew R, Goemann M, Hamper U, DeJong R, Detorie N, Rodriguez R, Haulk T, DeMarzo A M, Piantadosi S, Yu D C, Chen Y, Henderson D R, Carducci M A, Nelson W G, Simons J W. A phase I trial of CV706, "A replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy", Cancer Res 61(20):7464-72, 2001). The present inventors have also found that infecting cancer cells with a virus having a telomerase promoter and replication ability can kill the cancer cells by viral replication (Kawashima T, Kagawa S, Kobayashi N, Shirakiya Y, Umeoka T, Teraishi F, Taki M, Kyo S, Tanaka N, and Fujiwara T., Related Articles, Links Abstract "Telomerase-specific replication-selective virotherapy for human cancer", Clin Cancer Res 10(1):285-92, 2004).

[0005] However, in situ cancer detection system during surgical operation has not yet been developed because of the difficulty in targeting cancer cells. Further, no research has been known to date in which the living body is infected with a virus and the viral kinetics within cancer cells is actually applied to visualization of cancer tissues.

SUMMARY OF THE INVENTION

[0006] It is an object of the present invention to provide a reagent for detecting cancer cells or diagnosing cancers, and a cell death-inducing agent, which are capable of visualizing cancer cells not only in vitro but also in vivo. It is another object of the present invention to provide a method of treating or preventing cancer in a subject.

[0007] As a result of intensive and extensive researches toward the solution of the above problems, the present inventors have found that it is possible to detect cancer cells with extremely high sensitivity and even in vivo, by integrating a gene encoding a fluorescence labeling protein in E3 region of a viral genome and integrating a replication cassette comprising a human telomerase promoter, an E1A gene, an IRES sequence and an E1B gene in this order in E1 region, and then expressing both cassettes. Also, the present inventor have found that a recombinant virus comprising the replication cassette and the labeling cassette above has an anti-cancer effect. Thus, the present invention has been achieved.

[0008] The present invention relates to the following.

[0009] (1) A reagent for cancer cell detection, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome.

[0010] (2) A reagent for cancer diagnosis, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome.

[0011] In (1) and (2) above, these reagents may be used in in vivo detection, diagnosis or navigation surgery. As a specific example of the promoter from human telomerase, hTERT promoter may be given. As a specific example of the labeling protein, GFP may be given. As the promoter capable of regulating the expression of a gene encoding this labeling protein, a cytomegalovirus promoter or hTERT promoter may be used, for example. As the virus, an adenovirus may be used, for example.

[0012] (3) A cell death-inducing agent, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a cell death-inducing cassette comprising a gene encoding a protein associated with cell death induction and a promoter capable of regulating the expression of the gene encoding the protein associated with cell death induction is integrated in E3 region of the viral genome.

[0013] In this cell death-inducing agent, the promoter from human telomerase may be hTERT promoter. Examples of proteins associated with cell death induction include immunity-associated proteins, apoptosis-inducing proteins and telomerase-associated proteins. More specifically, PA28 may be given as an immunity-associated protein; TRAIL may be given as an apoptosis-inducing protein; and AU5 may be given as a telomerase-associated protein. The promoter capable of regulating the expression of a protein associated with cell death induction may be a cytomegalovirus promoter or hTERT promoter; and the virus may be an adenovirus. As a cell of the present invention, a cancer cell may be used.

[0014] (4) A pharmaceutical composition for the treatment of cancer in a subject, comprising a therapeutically effective amount of a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome, and a pharmaceutically acceptable carrier.

[0015] In the pharmaceutical composition of the present invention, the promoter from human telomerase may be hTERT promoter. As a specific example of the labeling protein, GFP may be given. As the promoter capable of regulating the expression of a gene encoding this labeling protein, a cytomegalovirus promoter may be used, for example. As the virus, an adenovirus may be used, for example.

(5) A method of detecting cancer cells, comprising infecting cancer cells with the reagent of (1) above and detecting the fluorescence emitted by the cancer cells.

(6) A method of cancer diagnosis, comprising infecting cancer cells with the reagent of (2) above and detecting the fluorescence emitted by the cancer cells.

(7) A method of inducing cell death in a target cell, comprising infecting the target cell with the cell death-inducing agent of (3) above.

[0016] According to the present invention, a reagent for detecting cancer cells or for diagnosing cancers, and a cell death-inducing agent are provided. Since the reagent of the present invention is capable of detecting cancer cells with extremely high sensitivity even in vivo, the reagent is useful in the so-called navigation surgery or the like.

[0017] (8) A method of treating or preventing cancer in a subject, comprising administering to the subject a therapeutically effective amount of a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome.

[0018] In the method of the present invention, the promoter from human telomerase may be hTERT promoter. As a specific example of the labeling protein, GFP may be given. As the promoter capable of regulating the expression of a gene encoding this labeling protein, a cytomegalovirus promoter may be used, for example. As the virus, an adenovirus may be used, for example.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a diagram showing the structure of Telomelysin-GFP.

[0020] FIG. 2 is a diagram showing the replication of the non-replicating virus.

[0021] FIG. 3 is a diagram showing the results of detection of cancer cells by in vitro co-infection with Ad-GFP.

[0022] FIG. 4 is a diagram showing the results of detection of human cancer tissues by in vivo co-infection with Ad-GFP.

[0023] FIG. 5 is a diagram showing the morphological changes in human lung cancer cells infected with Telomelysin-GFP.

[0024] FIG. 6 is a diagram showing the emission of GFP fluorescence in human lung cancer cells infected with Telomelysin-GFP.

[0025] FIG. 7 is a diagram showing the replication of Telomelysin-GFP determined by quantitative real time-PCR.

[0026] FIG. 8 is a diagram showing the emission of GFP fluorescence in human large colon cancer cells infected with Telomelysin-GFP.

[0027] FIG. 9 is a diagram showing the replication of Telomelysin-GFP determined by quantitative real time-PCR.

[0028] FIG. 10 is a diagram showing the morphological changes in normal human lung fibroblast cells (NHLF) infected with Telomelysin-GFP.

[0029] FIG. 11 is a diagram showing the emission of GFP fluorescence in normal human lung fibroblast cells (NHLF) infected with Telomelysin-GFP.

[0030] FIG. 12 is a diagram showing comparison of Telomelysin-GFP replications determined by quantitative real time-PCR.

[0031] FIG. 13 is a diagram showing the intratumoral proliferation/replication of Telomelysin-GFP observed by fluorescence imaging.

[0032] FIG. 14 is a diagram showing the intratumoral proliferation/replication of Telomelysin-GFP observed by fluorescence imaging.

[0033] FIG. 15 is a diagram showing the intratumoral proliferation/replication of Telomelysin-GFP in a lymph node metastasis model observed by fluorescence imaging.

[0034] FIG. 16 is a diagram showing histological analysis in an orthotopic rectal cancer model using nude mouse and HT29 human large colon cancer cells.

[0035] FIG. 17 is a diagram showing ventrotomy findings in an orthotopic rectal cancer model using nude mouse and HT29 human large colon cancer cells.

[0036] FIG. 18 is a diagram showing the intratumoral proliferation/replication of Telomelysin-GFP in HT29 rectal tumor and para-aortic lymph nodes observed by fluorescence imaging.

[0037] FIG. 19 is a diagram showing the intratumoral proliferation/replication of Telomelysin-GFP in para-aortic lymph nodes observed by fluorescence imaging.

[0038] FIG. 20 is a diagram showing the intratumoral proliferation/replication of Telomelysin-GFP in para-aortic lymph nodes observed by fluorescence imaging.

[0039] FIG. 21 is a diagram showing the morphological changes and the expression of GFP fluorescence in H1299 human lung cancer cells infected with OBP-401 (Telomelysin-GFP).

[0040] The morphological changes indicate a cell death of H1299 cells induced by OBP-401.

[0041] FIG. 22 is a diagram showing antitumor effects of intratumorally injected against established flank H1299 xenograft tumors in nu/nu mice. Tumor growth was expressed by tumor mean volume.+-.SE. Statistical significance was defined as p<0.05 (*) (Student's t-test). Arrows, day of treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0042] Hereinbelow, the present invention will be described in detail. The present invention will now be described with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0044] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

1. Reagent for Cancer Cell Detection and Detection Method

[0045] The present invention relates to a reagent for detecting cancer cells, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome. Further, the present invention relates to a method of detecting cancer cells, comprising infecting cancer cells with the reagent and detecting the fluorescence emitted by the cancer cells. In the present invention, the term "recombinant virus" means a virus in which the replication cassette and the labeling cassette described later are integrated in the genome. The virus used in the present invention is not particularly limited, but an adenovirus is preferable from the viewpoint of safety. Among adenovirus species, type 5 adenovirus is especially preferable mainly because it is easy to handle.

[0046] The recombinant virus used in the present invention has a replication cassette integrated in a region corresponding to E1 region of adenovirus genome and a labeling cassette integrated in a region corresponding to E3 region of adenovirus genome. The replication cassette comprises a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order. The E1A gene, IRES sequence and E1B gene are driven by the human telomerase promoter, which results in cancer cell-specific and telomerase-specific proliferation/replication of the virus. The labeling cassette comprises a promoter and a gene encoding a labeling protein. For example, the gene encoding the labeling protein is driven by a CMV (cytomegalovirus) promoter or hTERT promoter (FIG. 1).

[0047] The "telomerase promoter" determines the transcription initiation site for telomerase and directly regulates the frequency of transcription. Telomerase is an enzyme that maintains the length of telomeres, standing against the shortening of telomeres at the time of replication of eukaryotic chromosomes. The type of such telomerase promoter is not particularly limited, and any suitable telomerase promoter compatible with the virus to be used for the expression of the gene of interest may be used. For example, the promoter for human telomerase reverse transcriptase (hTERT) is preferable. A number of transcription factor-binding sequences are confirmed in a 1.4 kbp region upstream of the 5' end of hTERT gene. This region is believed to be hTERT promoter. In particular, a 181 bp sequence located upstream of the translation initiation site is a core region important for the expression of the downstream gene. In the present invention, any sequence comprising this core region may be used. Preferably, an upstream sequence of approximately 378 bp containing the entire core region is used as hTERT promoter. It has been confirmed that this sequence of approximately 378 bp is equivalent to the 181 bp core region alone in gene expression efficiency. The nucleotide sequence of an hTERT promoter of 455 bp is shown in SEQ ID NO: 1.

[0048] The nucleotide sequence of hTERT promoter is not limited to the sequence as shown in SEQ ID NO: 1. Nucleotide sequences of nucleotides which hybridize under stringent conditions to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of SEQ ID NO: 1 and have hTERT promoter activity may also be included as a sequence for hTERT promoter. Such nucleotides may be obtained from cDNA libraries or genomic libraries by known hybridization methods such as colony hybridization, plaque hybridization, Southern blotting, etc. using the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or a part thereof as a probe. cDNA libraries may be prepared according to the method described in Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)). Alternatively, commercial cDNA libraries or genomic libraries may be used.

[0049] In the above hybridization, examples of stringent conditions include 1.times.SSC to 2.times.SSC, 0.1% to 0.5% SDS and 42.degree. C. to 68.degree. C. More specifically, an example may be given where prehybridization is performed at 60-68.degree. C. for more than 30 minutes, and then washing is performed in 2.times.SSC, 0.1% SDS at room temperature for 5-15 minutes 4 to 6 times.

[0050] For detailed procedures of hybridization, see, for example, Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)), in particular, Section 9.47-9.58.

[0051] The reason why an E1A gene, an IRES sequence and an E1B gene are located in this order in the present invention is that insertion of the IRES sequence between the E1A gene and E1B gene will results in higher replication ability of the virus when a host cell has been infected with it. E1A gene and E1B gene are genes included in E1 gene. This is one of early genes of viruses, which have early (E) genes and late (L) genes involved in their DNA replication, and encodes a protein involved in the regulation of transcription of viral genome. E1A protein encoded by E1A gene activates the transcription of a group of genes (E1B, E2, E4, etc.) necessary for the production of infectious virus. E1B protein encoded by E1B gene assists the accumulation of late gene (L gene) mRNA in the cytoplasm of the infected host cell to thereby inhibit the protein synthesis in the host cell. Thus, E1B protein promotes viral replication. The nucleotide sequences of E1A gene and E1B gene are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0052] E1A and E1B may have, other than those nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively, nucleotide sequences which hybridize under stringent conditions to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of SEQ ID NO: 2 or SEQ ID NO: 3 and encode a protein having E1A or E1B activity. Such nucleotide sequences may be obtained from cDNA libraries or genomic libraries by known hybridization methods such as colony hybridization, plaque hybridization, Southern blotting, etc. using the polynucleotide, or a part thereof, consisting of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 as a probe. cDNA libraries may be prepared according to the method described in Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)). Alternatively, commercial cDNA libraries or genomic libraries may be used. In the above hybridization, examples of stringent conditions include 1.times.SSC to 2.times.SSC, 0.1% to 0.5% SDS and 42.degree. C. to 68.degree. C. More specifically, an example may be given where prehybridization is performed at 60-68.degree. C. for more than 30 minutes, and then washing is performed in 2.times.SSC, 0.1% SDS at room temperature for 5-15 minutes 4 to 6 times. For detailed procedures of hybridization, see, for example, Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)), in particular, Section 9.47-9.58.

[0053] IRES (Internal Ribosome Entry Site) is a protein synthesis initiation signal specific to picornavirus. It is believed that this site serves as a ribosome-binding site because it has a complementary sequence to the 3' terminal sequence of 18 S ribosomal RNA. It is known that mRNA derived from a virus which belongs to picornaviridae is translated via this sequence. Translation efficiency from IRES sequence is high. Even from the middle of mRNA, protein synthesis is performed in a cap structure non-dependent manner. Therefore, in the virus of the present invention, both E1A gene and E1B gene (which is located downstream of the IRES sequence) are translated independently by a human telomerase promoter. By using IRES, the control of expression by a telomerase promoter is exerted on E1A gene and E1B gene independently. Therefore, compared to cases where either E1A gene or E1B gene is controlled by a telomerase promoter, viral replication can be more strictly limited to those cells having telomerase activity. An IRES sequence is shown in SEQ ID NO: 4.

[0054] IRES may have, other than the nucleotide sequence as shown in SEQ ID NO: 4, nucleotide sequences which hybridize under stringent conditions to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of SEQ ID NO: 4 and encode a protein having IRES activity. Such nucleotide sequences may be obtained from cDNA libraries or genomic libraries by known hybridization methods such as colony hybridization, plaque hybridization, Southern blotting, etc. using the polynucleotide, or a part thereof, consisting of the nucleotide sequence of SEQ ID NO: 4 as a probe. cDNA libraries may be prepared according to the method described in Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)). Alternatively, commercial cDNA libraries or genomic libraries may be used. In the above hybridization, examples of stringent conditions include 1.times.SSC to 2.times.SSC, 0.1% to 0.5% SDS and 42.degree. C. to 68.degree. C. More specifically, an example may be given where prehybridization is performed at 60-68.degree. C. for more than 30 minutes, and then washing is performed in 2.times.SSC, 0.1% SDS at room temperature for 5-15 minutes 4 to 6 times. For detailed procedures of hybridization, see, for example, Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)), in particular, Section 9.47-9.58.

[0055] In the present invention, a promoter from human telomerase is located upstream of the E1 gene because such a promoter is capable of promoting the replication in cells having telomerase activity.

[0056] The genes contained in the replication cassette of the present invention may be obtained by conventional genetic engineering techniques. For example, as a genetic engineering technique, a method of nucleic acid synthesis with a commonly used DNA synthesizer may be used. Alternatively, after a genetic sequence to be used as a template is isolated or synthesized, primers specific to each gene may be designed and the genetic sequence may be amplified with a PCR apparatus (PCR method; Current Protocols in Molecular Biology, John Wiley & Sons (1987), Section 6.1-6.4); or a gene amplification method using a cloning vector may be used. One of ordinary skill in the art can readily carry out the above methods according to, for example, Moleculer Cloning 2.sup.nd Ed., Cold Spring Harbor Laboratory Press (1989). Purification of resultant PCR products may be performed by known methods such as a method using ethidium bromide, a method using SYBR Green I (Molecular Probes), a method with GENECLEAN (Funakoshi), QIAGEN (QIAGEN), etc. using agarose gel, a method using DEAE-cellulose filter, freeze & squeeze method or a method using a dialysis tube. When agarose gel is used, PCT products are electrophoresed on agarose gel and resultant DNA fragments are cut out from the gel and purified. If necessary, it is possible to confirm by conventional sequencing methods that an expected gene has been obtained. For example, dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) or the like may be used for this purpose. Alternatively, it is also possible to analyze the sequence with an appropriate DNA sequencer (e.g., ABI PRISM; Applied Biosystems).

[0057] Subsequently, individual genes thus obtained are ligated in a specific order. First, above genes are digested with known restriction enzymes, and the resultant DNA fragments are inserted into a known vector according to a known method for ligation. Specific examples of known vectors include pIRES vector which comprises the IRES (internal ribosome entry site in mRNA) of encephalomyocarditis virus (ECMV) and is capable of translating two open reading frames (ORFs) from one mRNA; Escherichia coli-derived plasmids (such as pCR4, pCR2, pCR2.1, pBR322, pBR325, pUC12 and pUC13); Bacillus subtilis-derived plasmids (such as pUB110, pTP5 and pC194); yeast-derived plasmids (such as pSH19 and pSH15); bacteriophages such as .lamda. phage; animal viruses such as retrovirus, vaccinia virus and baculovirus; pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo and so forth. In the present invention, use of pIRES vector is preferable. With this vector, it is possible to prepare a replication cassette containing "a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene" in this order by inserting necessary genes into the multi-cloning site in this order. DNA ligase may be used for ligation of DNAs. Hereinbelow, an example where hTERT is used as human telomerase in the present invention will be described specifically.

[0058] E1A gene and E1B gene may be amplified in E1 gene-expressing cells, such as 293 cells, by carrying out RT-PCR and/or DNA-PCR using primers such as E1A-S, E1A-AS, E1B-S and E1B-AS. If necessary, their sequences are confirmed using a known method such as TA cloning. Then, DNA fragments of E1A and E1B may be cut out using a known restriction enzyme.

[0059] Subsequently, a replication cassette consisting of hTERT-E1A-IRES-E1B to be used in the present invention may be prepared by inserting individual genes into a multi-cloning site or the like of a known vector (such as pIRES vector) to give the following order: "hTERT promoter sequence-E1A-IRES-E1B". Alternatively, if necessary, it is also possible to remove a cytomegalovirus (CMV) promoter from a known vector such as pShuttle with known restriction enzymes, and to insert into that site a sequence cut out from phTERT-E1A-IRES-E1B with appropriate restriction enzymes. The adenovirus in which only the replication cassette consisting of hTERT-E1A-IRES-E1B to be used in the present invention has been integrated is designated "Telomelysin". By expressing E1 gene necessary for proliferation of adenovirus under the control of hTERT promoter, it is possible to proliferate the virus in a cancer cell-specific manner.

[0060] In the recombinant virus used as a reagent of the present invention, a labeling cassette is also included together with the replication cassette. The "labeling cassette" is integrated in E3 region of the viral genome.

[0061] Here, it should be noted that the primary function of the virus vector used in the present invention is cytotoxicity by viral replication. Therefore, in order to use the reagent of the present invention for the purpose of diagnosing microcancer tissues, occurrence of the cytotoxicity is preferably as late as possible. This is because the emission of fluorescence caused by the replication of the recombinant virus of the present invention disappears when cells are destroyed, and it becomes difficult to identify the site of the microcancer tissue.

[0062] On the other hand, E3A and E3B exist in E3 region of adenovirus, and 11.6 kDa ADP (adenovirus death protein) in E3A region has a function to promote cytotoxicity and viral dispersion.

[0063] Therefore, in the recombinant virus used in the present invention, viral genomic regions encoding proteins with a function to promote cytotoxicity and viral dispersion (such ADP-encoding E3 region) are deleted to thereby delay the timing of cell death and facilitate identification of cancer cells by emission of GFP fluorescence or the like.

[0064] The labeling protein which constitutes the labeling cassette is a protein which emits light in those cells where the above-described virus has replicated, and is visualized. Preferably, a substance which emits fluorescence is used. Examples of such substances include, but are not limited to, green fluorescent protein (GFP) derived from luminescent jellyfish such as Aequorea victoria, enhanced-humanized GFP (EGFP) or red-shift GFP (rsGFP) which are modified variants of GFP (GFP variants). It is also possible to use yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), or Renilla reniformis-derived GFP. A gene encoding any of these proteins may be used in the present invention.

[0065] A promoter capable of regulating the expression of the above-described gene may be any promoter as long as it is compatible with the virus to be used for the expression of the above gene of interest. Specific examples of such promoters include, but are not limited to, cytomegalovirus (CMV) promoter, hTERT promoter, SV40 late promoter, MMTV LTR promoter, RSV LTR promoter and SR.alpha. promoter. Preferably, CMV promoter or hTERT promoter may be used.

[0066] The recombinant gene contained in the labeling cassette of the present invention may be obtained by conventional genetic engineering techniques. For example, as a genetic engineering technique, a method of nucleic acid synthesis with a commonly used DNA synthesizer may be used. Alternatively, after a genetic sequence to be used as a template is isolated or synthesized, primers specific to each gene may be designed and the genetic sequence may be amplified with a PCR apparatus (PCR method; Current Protocols in Molecular Biology, John Wiley & Sons (1987), Section 6.1-6.4); or a gene amplification method using a cloning vector may be used. One of ordinary skill in the art can readily carry out the above methods according to, for example, Molecular Cloning 2.sup.nd Ed., Cold Spring Harbor Laboratory Press (1989). Purification of the resultant PCR product may be performed by known methods such as a method using ethidium bromide, a method using SYBR Green I (Molecular Probes), a method with GENECLEAN (Funakoshi), QIAGEN (QIAGEN), etc. using agarose gel, a method using DEAE-cellulose filter, freeze & squeeze method or a method using a dialysis tube. When agarose gel is used, the PCT production is electrophoresed on agarose gel and resultant DNA fragments are cut out from the gel and purified. If necessary, it is possible to confirm by conventional sequencing methods that an expected gene has been obtained. For example, dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) or the like may be used for this purpose. Alternatively, it is also possible to analyze the sequence with an appropriate DNA sequencer (e.g., ABI PRISM; Applied Biosystems). Subsequently, the gene thus obtained is digested with known restriction enzymes. The recombinant gene is so designed that the resultant, digested DNA fragment (encoding the labeling protein) is located downstream of a gene fragment encoding the above-described promoter. Here, shuttle plasmid pHM11 or the like may be used as a plasmid. The two genes (for the labeling protein and the promoter) are ligated with DNA ligase and inserted into a vector to thereby prepare a recombinant gene for the labeling cassette.

[0067] As a known vector, pShuttle vector, Escherichia coli-derived plasmid (such as pCR4, pCR2, pCR2.1, pBR322, pBR325, pUC12 or pUC13); Bacillus subtilis-derived plasmid (such as pUB110, pTP5 or pC194); yeast-derived plasmid (such as pSH19 or pSH15); bacteriophage such as .lamda. phage; animal viruse such as retrovirus, vaccinia virus or baculovirus; pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo or the like may be used.

[0068] Subsequently, a recombinant gene comprising the above described replication cassette and labeling cassette is cut out with appropriate restriction enzymes and inserted into an appropriate virus expression vector, to thereby prepare a recombinant virus. Examples of virus expression vectors include adenovirus, retrovirus, vaccinia virus and baculovirus. As described above, adenovirus (in particular, type 5 adenovirus) is preferably used. For integration of the cassettes into virus, methods such as electroporation, the liposome method, the spheroplast method or the lithium acetate method may be used.

[0069] In the present invention, specifically, the recombinant gene may be prepared by inserting CMV-EGFP-SV40P (A) from pEGFP-N1 (CLONTECH) into shuttle plasmid pHM11, and inserting Csp45I fragment of this plasmid into ClaI site of pShuttle vector in which phTERT-E1A-IRES-E1B has been integrated.

[0070] In the present invention, specifically, a sequence of a necessary region may be cut out with restriction enzymes from the recombinant gene as prepared above, and inserted into a viral DNA such as Adeno-X Viral DNA using a commercial kit such as Adeno-X Expression System (CLONTECH) (the resultant product is designated "AdenoX-hAIB").

[0071] This AdenoX-hAIB is linearized with a known restriction enzyme and then transfected into cultured cells, such as 293 cells, to thereby prepare an infectious recombinant virus.

[0072] The target cancer cells to be detected, treated or prevented in the present invention are not limited. Cancer cells of any kind may be used. For example, solid cancers in the head and neck, stomach, large colon, lung, liver, prostate, pancreas, esophagus, bladder, gallbladder/bile duct, breast, uterus, thyroid, ovary, etc.; or leukemia, lymphoma, sarcoma, mesenchymal tumor or the like may be used. Most of cancer cells derived from human tissues show increase in telomerase activity. The present invention is capable of detecting those cancer cells in general where proliferation has been activated by such telomerase activity.

[0073] Since telomerase expression is extremely high in cancer cells compared to normal cells, hTERT is expressed in telomerase-containing cancer cells and the replication cassette functions therein. As a result, the virus replicates, which in turn increases the replication of the labeling protein. Thus, the labeling protein is expressed and visualized.

[0074] Therefore, when the reagent of the present invention does not emit fluorescence in normal cells, but whereas emits fluorescence in cancer cells. Thus, it is possible to observe cancer cells visually.

[0075] For infecting cells with a recombinant virus, the following method may be used, for example. First, cells such as human large colon cancer cell SW620, human lung cancer cells A549 and H1299 are plated in culture plates containing an appropriate culture broth and cultured in the presence of CO.sub.2 gas at 37.degree. C. As the culture broth, one which is conventionally used for culturing animal cells may be used, e.g. DMEM, MEM, or RPMI-1640. If necessary, serum, antibiotics, vitamins, or the like may be added thereto. By inoculating a specific amount (0.1-10 MOI (multiplicity of infection), preferably 1 MOI) of the recombinant virus of the present invention, the cultured cells are infected. MOI means a ratio between the viral quantity (infective unit) and the number of cells when a specific amount of cultured cells are infected with a specific amount of viral particles. MOI is used as an indicator when cells are infected with virus.

[0076] In order to confirm viral replication, cells infected with virus are recovered and DNA is extracted therefrom. Then, the DNA is subjected to real time-PCR using primers targeting an appropriate gene contained by the virus of the present invention. Thus, quantitative analysis is possible.

[0077] With respect to the detection of labeled cells, cancer cells can be visualized because cells where viral replication is observed emit a specific fluorescence (e.g. green fluorescence when GFP is used) by exposing to excitation light. For example, when cells infected with virus are observed under fluorescence microscope, emission of GFP fluorescence in the cells can be observed. For the observation of infected cells with the passage of time, emission of GFP fluorescence may be observed with a CCD camera.

[0078] For the real time labeling and detection of cells of interest in vivo, the recombinant virus of the present invention may be administered into the living body.

[0079] The reagent of the present invention may be applied to the diseased site as it is. Alternatively, the reagent of the present invention may be introduced into the living body (target cell or organ) by any known method, e.g. intravenous, intramuscular, intra-abdominal or subcutaneous injection; inhalation through the nasal cavity, oral cavity or lung; oral administration; intravascular administration using catheter or the like. Dose levels are selected appropriately depending on the kind of active ingredient, the administration route, the target of administration, and the age, body weight, sex, symptoms and other conditions of the patient. Usually, dose levels may be selected so that the virus of the present invention (the active ingredient) is administered at a daily dose of about 10.sup.6-10.sup.11 PFU (plaque forming units), preferably about 10.sup.9-10.sup.11 PFU. This amount may be administered once a day, or may be divided into several portions and administered at several times a day.

[0080] The reagent of the present invention makes it possible to observe the label in vivo in real time. Thus, the reagent of the present invention is advantageous for use as an in vivo diagnosis agent. This is useful in the so-called navigation surgery.

[0081] If excision is performed in a wide range including the diseased organ in a surgical operation, the patient who survived this surgical operation can enjoy a long survival. However, the rate of occurrence of complications caused by the surgical operation itself becomes high. Further, loss of the function of the excised organ inevitably influences on the daily life after the surgical operation. It is important in cancer treatment to introduce a low-invasive treatment to reduce the burden of patients while maintaining the remote result of long survival.

[0082] When a low-invasive operation is pursued by minimizing the area of excision, one of the information pieces wanted is the presence or absence of metastatic lymph nodes. As a method for obtaining that information, sentinel node (SN) is attracting attention. SN is the lymph node which first receives the lymph flow from tumors, and there is a hypothesis that the first micro-metastasis occurs in this lymph node. This hypothesis is called the SN theory. Although large scale clinical tests in breast cancer have already been started in primarily Europe and the United States, whether or not this theory is applicable to other solid tumors is still unknown. Examination has just been started.

[0083] In vivo cancer diagnosis system using the reagent of the present invention is capable of establishing the technology of allowing direct expression of a fluorescent protein in cancer cells and identifying tumor tissues or metastasis-positive lymph nodes by a highly sensitive, fluorescence detection system during surgical operation. In other words, the technology of "navigation surgery" can be established as a method that is more effective than SN. The recombinant virus of the present invention replicates in a great number of cancer cells having telomerase activity, and those cells can emit, for example, a strong green fluorescence of GFP.

[0084] From the analysis of mono-lymph node metastasis sites, about 10% of skip metastasis, i.e., incipient metastasis to the second group or more remote lymph nodes skipping over the first group lymph nodes has been reported. Based on this report, there are a large number of researchers who have pointed out the danger of SN navigation. However, the in vivo cancer diagnosis system using the reagent of the present invention identifies tumor tissues or metastasis-positive lymph nodes directly during the surgical operation in real time and the excision range is navigated. This system is original and epoch-making, and further it is extremely practical for smooth progress of surgical operation. Specifically, the reagent of the present invention is endoscopically injected into the site of tumor (e.g., gastric or large colon mucosa around gastric cancer or large colon cancer; internal region of tumors such as gastric cancer, large colon cancer, lung cancer, pancreatic cancer) several days prior to the surgical operation with the same manual technique used in SN navigation. Then, sufficient time is provided so that the virus is distributed into tumor-infiltrated tissues, metastatic tumor tissues or attending lymph nodes to replicate in tumor sites or metastasis-positive sites.

[0085] At the time of surgery, excitation light for GFP fluorescence is projected from the light source onto the surgery field after ventrotomy, and images from a special 3CCD camera are projected on a face mount display. By using a transmissible lens, the visual field of the actual surgical field can also be secured, and it is possible to detect metastasis-positive lymph nodes from overlapped GFP images. Further, by mounting a special filter, it becomes possible to recognize fluorescence with the eyes without using the camera.

2. Reagent for ex vivo Diagnosis

[0086] The reagent of the present invention is also applicable to an ex vivo diagnosis agent for the purpose of screening. Currently, quantitative determination of tumor markers is the most common method to know the presence of cancer which cannot be detected with the eyes or its primary focus cannot be identified. However, tumor markers are not necessarily satisfactory in their cancer specificity. Besides, it is extremely difficult to detect every cancer species with a single marker.

[0087] It has been confirmed that telomerase activity increases in 85% or more of human malignant tumors, and thus its cancer specificity is believed to be extremely high.

[0088] Ex vivo cancer diagnosis using the reagent of the present invention may be performed, for example, as described below.

[0089] Erythrocytes are removed from a total blood sample taken from a subject. To the remaining cell suspension liquid, the reagent of the present invention is added at a specific ratio (0.1-10 MOI, preferably 1 MOI) and mixed in a test tube. The mixture is left for a specific period of time (e.g., 12-48 hr) to promote infection of cancer cells with the virus and the resultant viral replication. Then, the GFP expression in the cell fraction is analyzed quantitatively by flow cytometry. With the use of this system, it becomes possible to detect free cancer cells present in the peripheral blood with high sensitivity. This method can be used for detecting free cancer cells present in the peripheral blood only in an extremely small quantity.

3. Cell Death-Inducing Agent and Method of Inducing Cell Death

[0090] The present invention provides a cell death-inducing agent, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a cell death-inducing cassette comprising a gene encoding a protein associated with cell death induction and a promoter capable of regulating the expression of the gene encoding the protein is integrated in E3 region of the viral genome. Preferably, the cell death-inducing agent of the present invention is used in gene therapy for cancers as an agent for inducing cell death in cancer cells, and may also be used for prevention of recurrence, inhibition and/or prevention of metastasis after surgical operation of cancers.

[0091] In the cell death-inducing cassette of the recombinant virus contained in the cell death-inducing agent of the present invention, a gene is integrated which is operated by a promoter and encodes a protein capable of inducing cell death.

[0092] In this cell death-inducing cassette used in the recombinant virus, a gene encoding a protein associated with cell death induction and a promoter capable of regulating the expression of the gene are contained. Therefore, when the cell death-inducing agent of the present invention is introduced into cancer cells, the virus replicates specifically in the cancer cells. As a result, intracellular expression level of the cell death-inducing protein increases, enabling induction of cell death only in the cancer cells without damaging normal cells.

[0093] A gene encoding a protein associated with cell death induction refers to a gene encoding a protein associated with the induction of cell death in a specific cell.

[0094] Specific examples of proteins associated with cell death induction include the following proteins, but are not limited to them. In the present invention, a gene encoding any of these proteins may be integrated.

[0095] As a specific example of immunity-associated protein, PA28 may be given. PA28 is a protein which activates intracellular proteasomes. When overexpressed, this protein causes immunological reactions and at the same time induces cell death. As a specific example of apoptosis-inducing protein, TRAIL may be given. TRAIL is a molecule which induces apoptotic cell death by binding to the receptor on cell surfaces. As a specific example of telomerase-associated protein, AU5 may be given. AU5 has a sequence capable of inducing cell death in cells having telomerase activity.

[0096] The genes for these proteins associated with cell death induction may be obtained by conventional genetic engineering techniques. For example, as a genetic engineering technique, a method of nucleic acid synthesis with a commonly used DNA synthesizer may be used. Alternatively, after a genetic sequence to be used as a template is isolated or synthesized, primers specific to each gene may be designed and the genetic sequence may be amplified with a PCR apparatus (PCR method; Current Protocols in Molecular Biology, John Wiley & Sons (1987), Section 6.1-6.4); or a gene amplification method using a cloning vector may be used. One of ordinary skill in the art can readily carry out the above methods according to, for example, Moleculer Cloning 2.sup.nd Ed., Cold Spring Harbor Laboratory Press (1989). Purification of the resultant PCR product may be performed by known methods such as a method using ethidium bromide, a method using SYBR Green I (Molecular Probes), a method with GENECLEAN (Funakoshi), QIAGEN (QIAGEN), etc. using agarose gel, a method using DEAE-cellulose filter, freeze & squeeze method or a method using a dialysis tube. When agarose gel is used, the PCT production is electrophoresed on agarose gel and resultant DNA fragments are cut out from the gel and purified. If necessary, it is possible to confirm by conventional sequencing methods that an expected gene has been obtained. For example, dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) or the like may be used for this purpose. Alternatively, it is also possible to analyze the sequence with an appropriate DNA sequencer (e.g., ABI PRISM; Applied Biosystems).

[0097] Antioncogenes are also included in cell death-inducing substances for cancer cells, because antioncogenes have a function of inhibiting the replication of cancer cells. For this purpose, the following antioncogenes used in conventional gene therapy may be enumerated.

[0098] p53 (SEQ ID NO: 11; Accession No. M14694): various kinds of cancers

[0099] p15 (SEQ ID NO: 12; Accession No. L36844): various kinds of cancers,

[0100] p16 (SEQ ID NO: 13; Accession No. L27211): various kinds of cancers

[0101] APC (SEQ ID NO: 14; Accession No. M74088): large colon cancer, gastric cancer, pancreatic cancer

[0102] BRCA-1 (SEQ ID NO: 15; Accession No. U14680): ovarian cancer, breast cancer

[0103] DPC-4 (SEQ ID NO: 16; Accession No. U44378): large colon cancer, pancreatic cancer

[0104] FHIT (SEQ ID NO: 17; Accession No. NM 112012): gastric cancer, lung cancer, uterus cancer

[0105] p73 (SEQ ID NO: 18; Accession No. Y11416): neuroblastoma

[0106] PATCHED (SEQ ID NO: 19; Accession No. U59464): basal cell carcinoma

[0107] Rbp 110 (SEQ ID NO: 20; Accession No. M15400): lung cancer, osteosarcoma

[0108] DCC (SEQ ID NO: 21; Accession No. X76132): large colon cancer

[0109] NF1 (SEQ ID NO: 22; Accession No. NM 000267): neurofibromatosis type 1

[0110] NF2 (SEQ ID NO: 23; Accession No. L11353): neurofibromatosis type 2

[0111] WT-1 (SEQ ID NO: 24; Accession No. NM 000378): Wilms' tumor

[0112] These antioncogenes may be obtained by conventional genetic engineering techniques. For example, as a genetic engineering technique, a method of nucleic acid synthesis with a commonly used DNA synthesizer may be used. Alternatively, after a genetic sequence to be used as a template is isolated or synthesized, primers specific to each gene may be designed and the genetic sequence may be amplified with a PCR apparatus (PCR method); or a gene amplification method using a cloning vector may be used. If necessary, it is possible to confirm by conventional sequencing methods that an expected gene has been obtained.

[0113] As a promoter capable of regulating the expression of the above gene, any promoter may be used as long as it is an appropriate promoter compatible with the virus to be used for the expression of the gene of interest. Preferably, a CMV promoter or hTERT promoter may be used. However, other promoters such as SV40 late promoter, MMTV LTR promoter, RSV LTR promoter and SR.alpha. promoter may also be used.

[0114] The cell death-inducing agent of the present invention may be applied to the diseased site as it is. Alternatively, the agent of the present invention may be introduced into the living body (target cell or organ) by any known method, e.g. intravenous, intramuscular, intra-abdominal, intranasal, intradermal or subcutaneous injection; inhalation through the nasal cavity, oral cavity or lung; oral administration; intravascular administration using catheter or the like; implantation (e.g., using slow release technology). Depending on the route of administration, the agent of the present invention may be required to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate it, such as those in the digestive tract.

[0115] The cell death-inducing agent of the present invention may be treated, for example, by the method such as freezing to enable easy handling and then used alone, or prepared into pharmaceutical compositions by mixing with known pharmaceutically acceptable carriers such as excipients, dispersions, fillers, binders, lubricants; or known additives (including such as buffers, isotonic agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, and sweetening agents). Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, or in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. Alternatively, the agent can be stored in lyophilized form to be rehydrated with an appropriate vehicle or carrier prior to use.

[0116] The cell death-inducing agent of the present invention may be administered orally or parenterally depending on the form of the agent, e.g. oral administration agents such as tablets, capsules, powders, granules, pills, liquids, syrups, sustained release formulations, etc. and parenteral administration agents such as injections, external medicines, suppositories, eye drops, etc. Preferably, local injection into muscle or abdominal cavity, or intravenous injection may be enumerated.

[0117] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be fluid to the extent that easy syringeability exists, unless the pharmaceutical form is a solid or semi-solid such as when slow release technology is employed. In any event, it must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms.

[0118] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents such as, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example, aluminum monostearate and gelatin.

[0119] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter-sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients selected from those enumerated above.

[0120] Dose levels are selected appropriately depending on the kind of active ingredient, the administration route, the target of administration, and the age, body weight, sex, symptoms and other conditions of the patient. Usually, dose levels may be selected so that the virus of the present invention (the active ingredient) is administered at a daily dose of about 10.sup.6-10.sup.11 PFU (plaque forming units), preferably about 10.sup.9-10.sup.11 PFU. This amount may be administered once a day, or may be divided into several portions and administered at several times a day.

[0121] When the virus of the present invention is administered, it is also possible to use a known immunosuppressant or the like to suppress the immunity of the living body to thereby make the viral infection easy.

[0122] Further, the virus of the present invention may be used jointly with at least one anticancer agent selected from the group consisting of known anticancer agents and radiation. Specific examples of anticancer agents include, but are not limited to, the following agents.

(1) Alkylating agents: These agents have an effect of causing cytotoxicity by introducing alkyl groups into the nucleic acid/protein of cancer cells. Examples include carboquone, busulphan (mustard drugs) and nimustine (nitrosoureas).

[0123] (2) Antimetabolic agents: These agents have an effect of inhibiting cell synthesis by antagonizing enzymes in metabolic processes. Examples include methotorexate (folates), mercaptopurine (purines), cytarabine (pyrimidines), fluorouracil, tegafur and carmofur.

(3) Antibiotics: These agents have an anti-cancer effect. Examples include actinomycin D, bleomycin, adriamycin and mitomycin C.

(4) Antimicrotubule agents: These agents act on microtubules and exhibit an anticancer effect. Examples include docetaxel, paclitaxel (taxanes) and vinorelbine, vincristine, vinblastine (alkaloids).

(5) Platinum preparations: These preparations have an effect of inhibiting DNA synthesis by forming intra- or inter-DNA strand crosslinks or DNA-protein crosslinks. Examples include cisplatin, carboplatin and nedaplatin.

[0124] (6) Topoisomerase inhibitors: Iinotecan (topoisomerase I inhibitor), podophyllotoxin derivatives (topoisomerase II inhibitor) and the like may be enumerated. Topoisomerase is an enzyme that catalyzes a reaction of changing the linking number of DNA by transiently cutting one or both strands of DNA.

[0125] It is believed that there is an extremely low possibility that the cell death-inducing agent of the present invention will produce side effects for the reasons described below. Thus, the cell death-inducing agent of the present invention can be a very safe preparation.

(1) There is little telomerase activity in normal somatic cells, and yet the virus of the present invention is hard to be infectious in suspending cells such as hematopoietic cells.

(2) Since the virus of the present invention has replication ability, it is possible to use this virus at a lower concentration than that of conventional non-replication competent virus used in conventional gene therapy.

(3) Even when the virus of the present invention has been administered in excess, antiviral action works through ordinary immune reaction in the living body.

[0126] It is possible to induce cell death in a target cell by infecting the target cell with the recombinant virus of the present invention. The kinds of target cells are not particularly limited. For example, tumor cells, cells with active replication, cells whose telomerase activity has been increased, or the like may be used.

[0127] The expressing "infecting cells with the recombinant virus" means as described above. In order to confirm whether cell death has been induced or not, morphological observation may be performed as described below. Briefly, cells adhering to the bottom of a culture dish are infected with the recombinant virus of the present invention. After a specific time period, the form of the cells becomes circular and they suspend in the culture broth as glossy cells peeled off from the bottom. At this point, the life maintaining mechanism in these cells has been broken up, and it can be measured that cell death has been induced. Alternatively, it is also possible to confirm cell death with a commercial viable cell assay kit using tetrazorium salts (e.g., MTT, XTT).

4. Pharmaceutical composition for the treatment of cancer in a subject and Method of treating or preventing cancer in a subject

[0128] The present invention provides a pharmaceutical composition for the treatment of cancer in a subject, comprising a therapeutically effective amount of a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome, and a pharmaceutically acceptable carrier.

[0129] Further, the present invention provides a method of treating or preventing cancer in a subject, comprising administering to the subject a therapeutically effective amount of the cell death-inducing agent or the pharmaceutical composition described above.

[0130] The term "cancer" has its understood meaning in the art, for example, an uncontrolled growth of tissue that has the potential to spread to distant sites of the body (i.e., metastasize). The term "tumor" is also understood in the art, for example, as an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. Preferably, the inventive methods disclosed herein are used to prevent and treat malignant tumors. The term "cancer", as used herein, includes but not limited to solid cancers in the head and neck, brain, kidney, stomach, large bowel, small intestine, colorectum, lung, liver, prostate, pancreas, esophagus, bladder, gallbladder/bile duct, squamous cell, breast, uterus, thyroid, ovary, bone, skin etc.; or leukemia, lymphoma, sarcoma, melanoma, carcinoma, mesenchymal tumor, neoplasm of the central nervous system (e.g., spinal axis tumors) or the like. Preferred are methods of treating and preventing tumor-forming cancers.

[0131] The term "subject", as used herein, includes humans, mouse, rat, rabbit, dog, cat cow, horse, and other organisms.

[0132] The pharmaceutical composition of the present invention may be applied to the diseased site as it is. Alternatively, the pharmaceutical composition of the present invention may be introduced into the living body (target cell or organ) by any known method, e.g. intravenous, intramuscular, intra-abdominal, intranasal, intradermal or subcutaneous injection; inhalation through the nasal cavity, oral cavity or lung; oral administration; intravascular administration using catheter or the like.

[0133] The virus included in the pharmaceutical composition of the present invention may be treated, for example, by the method such as freezing to enable easy handling and then used alone, or prepared into pharmaceutical compositions by mixing with known pharmaceutically acceptable carriers such as excipients, dispersions, fillers, binders; or known additives (including such as buffers, isotonic agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, and sweetening agents). Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, or in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. Alternatively, the agent can be stored in lyophilized form to be rehydrated with an appropriate vehicle or carrier prior to use.

[0134] The pharmaceutical composition of the present invention may be administered orally or parenterally depending on the form of the agent, e.g. oral administration agents such as tablets, capsules, powders, granules, pills, liquids, syrups, sustained release formulations, etc. and parenteral administration agents such as injections, external medicines, suppositories, eye drops, etc. Preferably, intratumoral injection, local injection into muscle or abdominal cavity, or intravenous injection may be employed.

[0135] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be fluid to the extent that easy syringeability exists, unless the pharmaceutical form is a solid or semi-solid such as when slow release technology is employed. In any event, it must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms.

[0136] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents such as, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example, aluminum monostearate and gelatin.

[0137] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter-sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients selected from those enumerated above.

[0138] Dose levels are selected appropriately depending on the kind of active ingredient, the administration route, the target of administration, and the age, body weight, sex, symptoms and other conditions of the patient. Usually, dose levels may be selected so that the virus of the present invention (the active ingredient) is administered at a daily dose of about 10.sup.6-10.sup.11 PFU (plaque forming units), preferably about 10.sup.9-10.sup.11 PFU. This amount may be administered once a day, or may be divided into several portions and administered at several times a day.

[0139] When the virus included in the pharmaceutical composition of the present invention is administered, it is also possible to use a known immunosuppressant or the like to suppress the immunity of the living body to thereby make the viral infection easy.

[0140] Further, the pharmaceutical composition of the present invention may be used jointly with at least one anticancer agent selected from the group consisting of known anticancer agents and radiation. Specific examples of anticancer agents include, but are not limited to, the following agents.

(1) Alkylating agents: These agents have an effect of causing cytotoxicity by introducing alkyl groups into the nucleic acid/protein of cancer cells. Examples include carboquone, busulphan (mustard drugs) and nimustine (nitrosoureas).

[0141] (2) Antimetabolic agents: These agents have an effect of inhibiting cell synthesis by antagonizing enzymes in metabolic processes. Examples include methotorexate (folates), mercaptopurine (purines), cytarabine (pyrimidines), fluorouracil, tegafur and carmofur.

(3) Antibiotics: These agents have an anti-cancer effect. Examples include actinomycin D, bleomycin, adriamycin and mitomycin C.

(4) Antimicrotubule agents: These agents act on microtubules and exhibit an anticancer effect. Examples include docetaxel, paclitaxel (taxanes) and vinorelbine, vincristine, vinblastine (alkaloids).

(5) Platinum preparations: These preparations have an effect of inhibiting DNA synthesis by forming intra- or inter-DNA strand crosslinks or DNA-protein crosslinks. Examples include cisplatin, carboplatin and nedaplatin.

[0142] (6) Topoisomerase inhibitors: Iinotecan (topoisomerase I inhibitor), podophyllotoxin derivatives (topoisomerase II inhibitor) and the like may be enumerated. Topoisomerase is an enzyme that catalyzes a reaction of changing the linking number of DNA by transiently cutting one or both strands of DNA.

[0143] It is believed that there is an extremely low possibility that the pharmaceutical composition of the present invention will produce side effects for the reasons described below. Thus, the pharmaceutical composition of the present invention can be a very safe preparation.

(1) There is little telomerase activity in normal somatic cells, and yet the virus included in the pharmaceutical composition of the present invention is hard to be infectious in suspending cells such as hematopoietic cells.

[0144] (2) Since the virus included in the pharmaceutical composition of the present invention has replication ability, it is possible to use this virus at a lower concentration than that of conventional non-replication competent virus used in conventional gene therapy.

(3) Even when the virus included in the pharmaceutical composition of the present invention has been administered in excess, antiviral action works through ordinary immune reaction in the living body.

[0145] Methods of preparing various pharmaceutical compositions with a specific amount of active compound are known, or will be apparent, to those skilled in the art.

[0146] Hereinbelow, the present invention will be described in more detail with reference to the following Examples. However, the present invention is not limited to these Examples.

EXAMPLE 1

Visualization of Cancer Cells by in vitro Co-Infection

[0147] This Example preliminary examined whether or not fluorescence will be emitted in vitro when cancer cells are co-infected with virus Telomelysin comprising the replication cassette and non-replicating virus Ad-GFP comprising the labeling cassette.

[0148] Human large colon cancer cell SW620 and human lung cancer cells A549 and H1299 were infected with 0.1 MOI (multiplicity of infection) of Ad-GFP (FIG. 2).

[0149] As a result, tendency to green color was hardly recognized when human large colon cancer cell SW620 and human lung cancer cells A549 and H1299 were infected with 0.1 MOI of Ad-GFP. However, when 1 MOI of TRAD was used jointly, fluorescence could be detected only in cancer cells, and no fluorescence was detected in normal cells such as human fibroblast cells WI38 and NHLF and human umbilical vascular endothelial cell (HUVEC) (FIG. 3).

EXAMPLE 2

Visualization of Cancer Tissues by in vivo Co-Infection with Telomelysin and Ad-GFP

[0150] This Example preliminary examined whether or not fluorescence will be emitted in vivo when cancer tissues are co-infected with virus Telomelysin comprising the replication cassette and non-replicating virus Ad-GFP comprising the labeling cassette.

[0151] Ad-GFP (8.times.10.sup.5 PFU) and TRAD (8.times.10.sup.6 PFU) were intratumorally administered to human large colon cancer SW5620 and human lung cancer A549 tumors transplanted subcutaneously into the dorsal of nude mice. Then, fluorescence was observed with the passage of time.

[0152] In any of the tumors, spot-like fluorescence had begun to be detected from day 2 after the administration and disappeared by day 14 (FIG. 4).

EXAMPLE 3

Detection of Cancer Cells with Telomelysin-GFP

1. Preparation of GFP-Expressing, Replication-Competent Virus (Telomelysin-GFP) which Comprises the Replication Cassette Comprising Telomerase Promoter and E1 Gene and the Labeling Cassette Comprising Gene Encoding GFP in a Single Virus

[0153] The outline of Telomelysin-GFP is shown in FIG. 1. Telomelysin-GFP proliferates/replicates cancer cell-specifically and telomerase-specifically because E1A/IRES/E1B is operated by the hTERT promoter. Further, Telomelysin-GFP also has an Aequorea victoria-derived GFP gene integrated in its E3 region which is operated by a promoter. Therefore, cells where viral replication is observed emit green fluorescence when excitation light is applied, which enables visualization of cancer cells.

[0154] Such a replication-incompetent virus was prepared as described below.

2. Preparation of Recombinant Virus

[0155] From RNA extracted from 293 cells, E1A gene of 897 bp was amplified by RT-PCR using the specific primers (E1A-S and E1A-AS) and PCR conditions described below. TABLE-US-00001 E1A-S: 5'-ACA CCG GGA CTG AAA ATG AG-3' (SEQ ID NO: 5) E1A-AS: 5'-CAC AGG TTT ACA CCT TAT GGC-3' (SEQ ID NO: 6)

Composition of the PCR solution: 1.times.PCR buffer [0156] 0.2 mM each dNTPs [0157] 5 mM MgCl.sub.2 [0158] 2.5 U AmpliTaq Gold [0159] 0.2 .mu.M each Primers Reaction conditions: 95.degree. C., 10 min [0160] (95.degree. C., 1 min; 56.degree. C., 1 min; 72.degree. C., 1.5 min).times.32 cycles [0161] 72.degree. C., 7 min [0162] 4.degree. C., 5 min

[0163] From DNA extracted from 293 cells, E1B gene of 1822 bp was amplified by DNA-PCR using the following primers E1B-S and E1B-AS. TABLE-US-00002 E1B-S: 5'-CTG ACC TCA TGG AGG CTT GG-3' (SEQ ID NO: 7) E1B-AS: 5'-GCC CAC ACA TTT CAG TAC CTC-3' (SEQ ID NO: 8)

[0164] The composition of the PCR solution and the reaction conditions (cycles, temperature) used were the same as used for the amplification of E1A gene.

[0165] Each PCR product was subjected to TA cloning (TA Cloning Kit Dual Promoter; Invitrogen) to thereby confirm their sequences. Then, DNA fragments of 911 bp (E1A) and 1836 bp (E1B) were cut out, respectively, with restriction enzyme EcoRI.

[0166] E1A and E1B were inserted into the MluI site and the SalI site, respectively, of pIRES vector (CLONTECH) in the normal orientation (E1A-IRES-E1B).

[0167] A 455 bp hTERT promoter sequence which had been cut out with restriction enzymes MluI and BglII was inserted into the XhoI site located upstream of the E1A of E1A-IRES-E1B in the normal orientation (phTERT-E1A-IRES-E1B).

[0168] The cytomegalovirus (CMV) promoter contained in pShuttle vector was removed by treatment with restriction enzymes MfeI and NheI. Then, a 3828 bp sequence cut out from phTERT-E1A-IRES-E1B using restriction enzymes NheI and NotI was inserted into that site (pSh-hAIB).

[0169] pEGFP-N1 (CLONTECH) was digested with AgeI/NheI, blunt-ended with Klenow fragment and self-ligated (pEGFP-N2).

[0170] This pEGFP-N2 was digested with NsiI/AflII and blunt-ended with T4 DNA polymerase, followed by preparation of a BglII site using BglII linker. This BglII fragment was inserted at the BamHI site of pHM11 (PHM11-EGFP-N2).

[0171] Further, Csp45I fragment from pHM11-EGFP-N2 was inserted at the ClaI site of pShuttle vector in which phTERT-E1A-IRES-E1B had been integrated (pSh-hAIB).

[0172] A 4381 bp sequence was cut out from the thus prepared recombinant gene (pSh-hAIB) using restriction enzymes I-CeuI and PI-SceI, and inserted into the Adeno-X Viral DNA of Adeno-X Expression System (CLONTECH) (AdenoX-hAIB). This AdenoX-hAIB was treated with restriction enzyme PacI for linearization and then transfected into 293 cells to thereby prepare an infectious recombinant adenovirus (hereinafter, referred to as "Telomelysin-GFP" or "OBP-401").

EXAMPLE 4

Detection Test on Human Lung Cancer Cells

1. Morphological Changes in Human Lung Cancer Cells Caused by Infection with Telomelysin-GFP

[0173] Human non-small-cell lung cancer-derived H1299 cells cultured in vitro were infected with Telomelysin-GFP at 1 MOI or 10 MOI. Specifically, H1299 cells were plated in 24-well plates at 5.times.10.sup.4 cells/well. After 24 hours, cells were counted and the virus was added to the culture broth to give a concentration of 1 MOI or 10 MOI. Subsequently, the morphology of cells was observed under inversed microscope with the passage of time to examine the cytotoxic activity of the virus.

[0174] As a result, cell death was induced by viral replication in a concentration dependent manner and also a time dependent manner. 120 hours after 10 MOI infection, most of the cells became circular and were suspending under inverted microscope (FIG. 5).

2. Emission of GFP Fluorescence in Human Lung Cancer Cells Caused by Infection with Telomelysin-GFP

[0175] The inverted microscopic images shown in FIG. 6 were observed under fluorescent microscope. The green fluorescence of GFP indicating viral replication in a concentration dependent manner and also a time dependent manner was observed (FIG. 6). 72 hours after 10 MOI infection, GFP expression was observed in the maximum number of cells. Then, the number of GFP-positive cells decreased as cell death was induced (FIG. 6).

3. Verification of Telomelysin-GFP Replication by Quantitative Real Time PCR

[0176] Human lung cancer cell H1299 was infected with Telomelysin-GFP at 10 MOI. Cell samples were harvested at 2, 26, 50 and 74 hours after the infection and DNA was extracted therefrom. Real time PCR was performed using the following primers targeting the E1A gene of Telomelysin-GFP, to thereby quantitatively analyze the viral proliferation/replication. The primers and PCR conditions used are as described below. TABLE-US-00003 E1A-S: 5'-CCT GTG TCT AGA GAA TGC AA-3' (SEQ ID NO: 9) E1A-AS: 5'-ACA GCT CAA GTC CAA AGG TT-3' (SEQ ID NO: 10)

Composition of PCR Solution: 1.times.LC FastStart DNA Master SYBR Green I [0177] 3 mM MgCl.sub.2 [0178] 0.5 .mu.M each Primer Reaction Conditions: 95.degree. C., 10 min [0179] (95.degree. C., 10 sec; 60.degree. C., 15 sec; 72.degree. C., 8 sec).times.40 cycles [0180] 70.degree. C., 15 sec [0181] 40.degree. C., 30 sec

[0182] The results revealed that Telomelysin-GFP had already replicated 1,000,000-fold at 26 hours after the infection (FIG. 7). Thereafter, the replication reached plateau, but GFP fluorescence was also enhanced gradually slightly after the replication (FIG. 7).

EXAMPLE 5

Detection Test on Human Large Bowel Cancer Cells

1. Emission of GFP Fluorescence in Human Large Bowel Cancer Cells Caused by Infection with Telomelysin-GFP

[0183] Human large colon cancer-derived SW620 cells were infected with Telomelysin-GFP at 10 MOI. Changes in the cells were observed with the passage of time under inverted microscope and fluorescent microscope.

[0184] As a result, GFP green fluorescence indicating viral replication in a time dependent manner was recognized as in the case of H1299 cells (FIG. 8).

2. Verification of Telomelysin-GFP Replication by Quantitative Real Time PCR

[0185] In the same manner as in H1299 cells, SW620 human large colon cancer cells were infected with Telomelysin-GFP at 10 MOI. Cell samples were harvested at 2, 26, 50, 74 and 98 hours after the infection and DNA was extracted therefrom. Then, viral replication was quantitatively analyzed by real time PCR. Real time PCR was performed using the following primers targeting the E1A gene of Telomelysin-GFP, to thereby quantitatively analyze the viral replication. Conditions of the real time PCR (composition of the reaction solution, cycle, time period, etc.) were the same as in H1299 cells.

[0186] The results revealed that Telomelysin-GFP had already replicated 1,000,000-fold at 26 hours after the infection and was almost plateau up to 98 hours after the infection (FIG. 9).

EXAMPLE 6

1. Morphological Changes in Human Normal Lung Fibroblast Cells (NHLF) Caused by Infection with Telomelysin-GFP

[0187] Normal human lung fibroblast cells (NHLF) cultured in vitro were infected with Telomelysin-GFP at 1 MOI or 10 MOI. Changes were observed under inverted microscope up to 120 hours after the infection.

[0188] As a result, no morphological changes were observed, and cell death was not induced (FIG. 10).

2. Emission of GFP Fluorescence in Normal Human Lung Fibroblast Cells Caused by Infection with Telomelysin-GFP

[0189] When the inverted microscopic images shown in FIG. 10 are observed under fluorescent microscope, emission of GFP fluorescence was observed in some cells. However, considering the cell density, the emission was extremely rare compared to that in cancer cells. Therefore, it was believed that Telomelysin-GFP hardly proliferates/replicates in normal cells (FIG. 11).

3. Verification of Telomelysin-GFP Replication by Quantitative Real Time PCR

[0190] H1299 human lung cancer cell, SW620 human large colon cancer cell, and normal human lung fibroblast cell (NHLF) were infected with Telomelysin-GFP at 10 MOI as described above. Cell samples were harvested with passage of time, and DNA was extracted therefrom. Then, viral replication was quantitatively analyzed by real time PCR.

[0191] The results revealed that Telomelysin-GFP had already replicated about 1,000,000-fold in cancer cells at 24 hours after the infection, and emitted remarkable GFP fluorescence at 72 hours after the infection (FIG. 12). On the other hand, replication was only about 1000-fold in NHLF cells even at 72 hours after the infection, and little GFP fluorescence could be detected (FIG. 12).

EXAMPLE 7

Detection of Intratumoral Proliferation/Replication of Telomelysin-GFP by Fluorescence Imaging

1. Telomelysin-GFP (10.sup.7 PFU) was administered into the tumor of H1299 human lung cancer transplanted into nude mice. Then, emission of GFP fluorescence was observed with a CCD camera with the passage of time.

[0192] The results revealed that emission of GFP fluorescence caused by Telomelysin-GFP replication began to be recognized within 24 hours after the infection, and that the range and luminance were gradually enhanced 3 days and 5 days after the infection (FIG. 13).

[0193] 2. In the same manner as described above, Telomelysin-GFP (10.sup.7 PFU) was administered into the tumor of H1299 human lung cancer transplanted into nude mice. One week and three weeks after the infection, subcutaneous tumor was removed. Emission of GFP fluorescence was observed on the entire tumor and on a cut surface using a CCD camera.

[0194] As a result, even when fluorescence emission was weak on the surface of the removed tumor, replication of Telomelysin-GFP could be confirmed in a wide range on the cut surface (FIG. 14). In tissues three weeks after the infection fluorescence was recognized on almost all over the tumor (FIG. 14).

[0195] 3. In the same manner as described above, HT29 human large colon cancer cell was transplanted into the rectal wall of nude mice as an orthotopic model, and Telomelysin-GFP (10.sup.7 PFU) was administered at the time when gross tumor was formed. Emission of GFP fluorescence caused by Telomelysin-GFP replication began to be recognized one week after the infection with a CCD camera (FIG. 15). The fluorescence emission was maintained even three weeks after the infection (FIG. 15).

EXAMPLE 8

1. Histological Analysis of Orthotopic Rectal Cancer Model Using Nude Mouse and HT29 Human Large Bowel Cancer Cell

[0196] HT29 human large colon cancer cell was transplanted into the rectal wall of nude mouse. When gross tumor was formed, the tumor was removed and analyzed after hematoxylin-eosin (HE) staining.

[0197] As a result, tumor was formed around the rectum, and tumor cell mass could be confirmed in lymph vessels in the rectal wall (FIG. 16).

2. Ventrotomy Findings in Orthotopic Rectal Cancer Model Using Nude Mouse and HT29 Human Large Bowel Cancer Cell

[0198] HT29 human large colon cancer cell was transplanted into the rectal wall, and ventrotomy was performed when gross tumor was formed. As a result, swelling was recognized in three lymph nodes (LN) around the aorta (FIG. 17)

3. Detection of Intratumoral Proliferation/Replication of Telomelysin-GFP in HT29 Rectal Tumor and Para-Aortic Lymph Nodes by Fluorescence Imaging

[0199] Emission of GFP fluorescence was recognized by fluorescence imaging with a CCD camera, in the transplanted HT29 rectal tumor and one of the three para-aortic lymph nodes (FIG. 18).

4. Detection of Intratumoral Proliferation/Replication of Telomelysin-GFP in Para-Aortic Lymph Nodes by Fluorescence Imaging

[0200] Emission of GFP fluorescence was recognized by fluorescence imaging with a CCD camera, in only one of the three para-aortic lymph nodes (FIG. 19).

5. Detection of Intratumoral Proliferation/Replication of Telomelysin-GFP in Para-Aortic Lymph Nodes by Fluorescence Imaging

[0201] Histological analysis of para-aortic lymph nodes detected metastatic tumor tissue in the only one lymph node which was found GFP fluorescence-positive by fluorescence imaging with a CCD camera. Thus, it was confirmed that Telomelysin-GFP replicates only in metastasis-positive lymph nodes (FIG. 20).

EXAMPLE 9

Morphological Changes and Expression of GFP Fluorescence in Human Lung Cancer Cells Caused by Infection with OBP-401 (Telomelysin-GFP)

[0202] Human non-small-cell lung cancer-derived H1299 cells cultured in vitro were infected with OBP-401 at an MOI of 10. Cell morphology was evaluated at indicated time points by phase-contrast photomicrography. Cells were also assessed for GFP expression under fluorescence microscopy (.times.200 magnification).

[0203] As a result, H1299 cells expressed bright GFP fluorescence as early as 20 hours after OBP-401 infection. The fluorescence intensity gradually increased followed by rapid cell death due to the cytopathic effect (CPE) of OBP-401, as evidenced by floating, highly light-refractile cells under phase-contrast photomicrographs (FIG. 21).

EXAMPLE 10

Antitumor Effects of Intratumorally Injected Against Established Flank H1299 Xenograft Tumors in nu/nu Mice

[0204] H1299 tumor cells (1.times.10.sup.7 cells/each) were subcutaneously injected into the right flank of mice. OBP-401 (1.times.10.sup.7 PFU/body) was administered intratumorally for 3 cycles every 2 days. PBS was used as a control. Six mice were used for each group. Tumor growth was expressed by tumor mean volume.+-.SE. Statistical significance was defined as p<0.05 (*) (Student's t-test).

[0205] As a result, administration of OBP-401 resulted in a significant tumor growth suppression compared with mock-treated tumors at 27 days after initiation of treatment (p<0.05) (FIG. 22).

REFERENCES

[0206] Reid T, Galanis E, Abbruzzese J, Sze D, Wein L M, Andrews J, Randlev B, Heise C, Uprichard M, Hatfield M, Rome L, Rubin J, Kim D. Hepatic arterial infusion of a replication-selective oncolytic adenovirus (d11520): phase II viral, immunologic, and clinical endpoints. Cancer Res 62 (21): 6070-9, 2002.

INDUSTRIAL APPLICABILITY

[0207] According to the present invention, a reagent for detecting cancer cells or diagnosing cancers and a cell death-inducing agent are provided. Since the reagent of the present invention is capable of detecting cancer cells with extremely high sensitivity even in the living body, the reagent is useful for the so-called navigation surgery or the like.

SEQUENCE LISTING FREE TEXT

[0208] SEQ ID NO: 5: Primer [0209] SEQ ID NO: 6: Primer [0210] SEQ ID NO: 7: Primer [0211] SEQ ID NO: 8: Primer [0212] SEQ ID NO: 9: Primer [0213] SEQ ID NO: 10: Primer

Sequence CWU 1

1

24 1 455 DNA homo sapiens 1 tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60 ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120 agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180 ttcgcgggca cagacgccca ggaccgcgct ccccacgtgg cggagggact ggggacccgg 240 gcacccgtcc tgccccttca ccttccagct ccgcctcctc cgcgcggacc ccgccccgtc 300 ccgacccctc ccgggtcccc ggcccagccc cctccgggcc ctcccagccc ctccccttcc 360 tttccgcggc cccgccctct cctcgcggcg cgagtttcag gcagcgctgc gtcctgctgc 420 gcacgtggga agccctggcc ccggccaccc ccgcg 455 2 899 DNA adenovirus 2 acaccgggac tgaaaatgag acatattatc tgccacggag gtgttattac cgaagaaatg 60 gccgccagtc ttttggacca gctgatcgaa gaggtactgg ctgataatct tccacctcct 120 agccattttg aaccacctac ccttcacgaa ctgtatgatt tagacgtgac ggcccccgaa 180 gatcccaacg aggaggcggt ttcgcagatt tttcccgact ctgtaatgtt ggcggtgcag 240 gaagggattg acttactcac ttttccgccg gcgcccggtt ctccggagcc gcctcacctt 300 tcccggcagc ccgagcagcc ggagcagaga gccttgggtc cggtttctat gccaaacctt 360 gtaccggagg tgatcgatct tacctgccac gaggctggct ttccacccag tgacgacgag 420 gatgaagagg gtgaggagtt tgtgttagat tatgtggagc accccgggca cggttgcagg 480 tcttgtcatt atcaccggag gaatacgggg gacccagata ttatgtgttc gctttgctat 540 atgaggacct gtggcatgtt tgtctacagt cctgtgtctg aacctgagcc tgagcccgag 600 ccagaaccgg agcctgcaag acctacccgc cgtcctaaaa tggcgcctgc tatcctgaga 660 cgcccgacat cacctgtgtc tagagaatgc aatagtagta cggatagctg tgactccggt 720 ccttctaaca cacctcctga gatacacccg gtggtcccgc tgtgccccat taaaccagtt 780 gccgtgagag ttggtgggcg tcgccaggct gtggaatgta tcgaggactt gcttaacgag 840 cctgggcaac ctttggactt gagctgtaaa cgccccaggc cataaggtgt aaacctgtg 899 3 1823 DNA adenovirus 3 ctgacctcat ggaggcttgg gagtgtttgg aagatttttc tgctgtgcgt aacttgctgg 60 aacagagctc taacagtacc tcttggtttt ggaggtttct gtggggctca tcccaggcaa 120 agttagtctg cagaattaag gaggattaca agtgggaatt tgaagagctt ttgaaatcct 180 gtggtgagct gtttgattct ttgaatctgg gtcaccaggc gcttttccaa gagaaggtca 240 tcaagacttt ggatttttcc acaccggggc gcgctgcggc tgctgttgct tttttgagtt 300 ttataaagga taaatggagc gaagaaaccc atctgagcgg ggggtacctg ctggattttc 360 tggccatgca tctgtggaga gcggttgtga gacacaagaa tcgcctgcta ctgttgtctt 420 ccgtccgccc ggcgataata ccgacggagg agcagcagca gcagcaggag gaagccaggc 480 ggcggcggca ggagcagagc ccatggaacc cgagagccgg cctggaccct cgggaatgaa 540 tgttgtacag gtggctgaac tgtatccaga actgagacgc attttgacaa ttacagagga 600 tgggcagggg ctaaaggggg taaagaggga gcggggggct tgtgaggcta cagaggaggc 660 taggaatcta gcttttagct taatgaccag acaccgtcct gagtgtatta cttttcaaca 720 gatcaaggat aattgcgcta atgagcttga tctgctggcg cagaagtatt ccatagagca 780 gctgaccact tactggctgc agccagggga tgattttgag gaggctatta gggtatatgc 840 aaaggtggca cttaggccag attgcaagta caagatcagc aaacttgtaa atatcaggaa 900 ttgttgctac atttctggga acggggccga ggtggagata gatacggagg atagggtggc 960 ctttagatgt agcatgataa atatgtggcc gggggtgctt ggcatggacg gggtggttat 1020 tatgaatgta aggtttactg gccccaattt tagcggtacg gttttcctgg ccaataccaa 1080 ccttatccta cacggtgtaa gcttctatgg gtttaacaat acctgtgtgg aagcctggac 1140 cgatgtaagg gttcggggct gtgcctttta ctgctgctgg aagggggtgg tgtgtcgccc 1200 caaaagcagg gcttcaatta agaaatgcct ctttgaaagg tgtaccttgg gtatcctgtc 1260 tgagggtaac tccagggtgc gccacaatgt ggcctccgac tgtggttgct tcatgctagt 1320 gaaaagcgtg gctgtgatta agcataacat ggtatgtggc aactgcgagg acagggcctc 1380 tcagatgctg acctgctcgg acggcaactg tcacctgctg aagaccattc acgtagccag 1440 ccactctcgc aaggcctggc cagtgtttga gcataacata ctgacccgct gttccttgca 1500 tttgggtaac aggagggggg tgttcctacc ttaccaatgc aatttgagtc acactaagat 1560 attgcttgag cccgagagca tgtccaaggt gaacctgaac ggggtgtttg acatgaccat 1620 gaagatctgg aaggtgctga ggtacgatga gacccgcacc aggtgcagac cctgcgagtg 1680 tggcggtaaa catattagga accagcctgt gatgctggat gtgaccgagg agctgaggcc 1740 cgatcacttg gtgctggcct gcacccgcgc tgagtttggc tctagcgatg aagatacaga 1800 ttgaggtact gaaatgtgtg ggc 1823 4 605 DNA adenovirus 4 tgcatctagg gcggccaatt ccgcccctct ccctcccccc cccctaacgt tactggccga 60 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt gattttccac catattgccg 120 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg 180 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt 240 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac 300 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca 360 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg 420 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg 480 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa 540 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataagct 600 tgcca 605 5 20 DNA Artificial primer 5 acaccgggac tgaaaatgag 20 6 21 DNA Artificial primer 6 cacaggttta caccttatgg c 21 7 20 DNA Artificial primer 7 ctgacctcat ggaggcttgg 20 8 21 DNA Artificial primer 8 gcccacacat ttcagtacctc 21 9 20 DNA Artificial primer 9 cctgtgtcta gagaatgcaa 20 10 20 DNA Artificial primer 10 acagctcaag tccaaaggtt 20 11 1307 DNA Homo sapiens 11 accgtccagg gagcaggtag ctgctgggct ccggggacac tttgcgttcg ggctgggagc 60 gtgctttcca cgacggtgac acgcttccct ggattggcag ccagactgcc ttccgggtca 120 ctgccatgga ggagccgcag tcagatccta gcgtcgagcc ccctctgagt caggaaacat 180 tttcagacct atggaaacta cttcctgaaa acaacgttct gtcccccttg ccgtcccaag 240 caatggatga tttgatgctg tccccggacg atattgaaca atggttcact gaagacccag 300 gtccagatga agctcccaga atgccagagg ctgctccccg cgtggcccct gcaccagcga 360 ctcctacacc ggcggcccct gcaccagccc cctcctggcc cctgtcatct tctgtccctt 420 cccagaaaac ctaccagggc agctacggtt tccgtctggg cttcttgcat tctgggacag 480 ccaagtctgt gacttgcacg tactcccctg ccctcaacaa gatgttttgc caactggcca 540 agacctgccc tgtgcagctg tgggttgatt ccacaccccc gcccggcacc cgcgtccgcg 600 ccatggccat ctacaagcag tcacagcaca tgacggaggt tgtgaggcgc tgcccccacc 660 atgagcgctg ctcagatagc gatggtctgg cccctcctca gcatcttatc cgagtggaag 720 gaaatttgcg tgtggagtat ttggatgaca gaaacacttt tcgacatagt gtggtggtgc 780 cctatgagcc gcctgaggtt ggctctgact gtaccaccat ccactacaac tacatgtgta 840 acagttcctg catgggcggc atgaaccgga ggcccatcct caccatcatc acactggaag 900 actccagtgg taatctactg ggacggaaca gctttgaggt gcgtgtttgt gcctgtcctg 960 ggagagaccg gcgcacagag gaagagaatc tccgcaagaa aggggagcct caccacgagc 1020 tgcccccagg gagcactaag cgagcactgc ccaacaacac cagctcctct ccccagccaa 1080 agaagaaacc actggatgga gaatatttca cccttcagat ccgtgggcgt gagcgcttcg 1140 agatgttccg agagctgaat gaggccttgg aactcaagga tgcccaggct gggaaggagc 1200 caggggggag cagggctcac tccagccacc tgaagtccaa aaagggtcag tctacctccc 1260 gccataaaaa actcatgttc aagacagaag ggcctgactc agactga 1307 12 837 DNA Homo sapiens 12 gaggactccg cgacggtccg caccctgcgg ccagagcggc tttgagctcg gctgcttccg 60 cgctaggcgc tttttcccag aagcaatcca ggcgcgcccg ctggttcttg agcgccagga 120 aaagcccgga gctaacgacc ggccgctcgg cactgcacgg ggccccaagc cgcagaagaa 180 ggacgacggg agggtaatga agctgagccc aggtctccta ggaaggagag agtgcgccgg 240 agcagcgtgg gaaagaaggg aagagtgtcg ttaagtttac ggccaacggt ggattatccg 300 ggccgctgcg cgtctggggg ctgcggaatg cgcgaggaga acaagggcat gcccagtggg 360 ggcggcagcg atgagggtct ggccacgccg gcgcggggac tagtggagaa ggtgcgacac 420 tcctgggaag ccggcgcgga tcccaacgga gtcaaccgtt tcgggaggcg cgcgatccag 480 gtcatgatga tgggcagcgc ccgcgtggcg gagctgctgc tgctccacgg cgcggagccc 540 aactgcgcag accctgccac tctcacccga ccggtgcatg atgctgcccg ggagggcttc 600 ctggacacgc tggtggtgct gcaccgggcc ggggcgcggc tggacgtgcg cgatgcctgg 660 ggtcgtctgc ccgtggactt ggccgaggag cggggccacc gcgacgttgc agggtacctg 720 cgcacagcca cgggggactg acgccaggtt ccccagccgc ccacaacgac tttattttct 780 tacccaattt cccaccccca cccacctaat tcgatgaagg ctgccaacgg ggagcgg 837 13 987 DNA Homo sapiens 13 cggagagggg gagaacagac aacgggcggc ggggagcagc atggagccgg cggcggggag 60 cagcatggag ccttcggctg actggctggc cacggccgcg gcccggggtc gggtagagga 120 ggtgcgggcg ctgctggagg cgggggcgct gcccaacgca ccgaatagtt acggtcggag 180 gccgatccag gtcatgatga tgggcagcgc ccgagtggcg gagctgctgc tgctccacgg 240 cgcggagccc aactgcgccg accccgccac tctcacccga cccgtgcacg acgctgcccg 300 ggagggcttc ctggacacgc tggtggtgct gcaccgggcc ggggcgcggc tggacgtgcg 360 cgatgcctgg ggccgtctgc ccgtggacct ggctgaggag ctgggccatc gcgatgtcgc 420 acggtacctg cgcgcggctg cggggggcac cagaggcagt aaccatgccc gcatagatgc 480 cgcggaaggt ccctcagaca tccccgattg aaagaaccag agaggctctg agaaacctcg 540 ggaaacttag atcatcagtc accgaaggtc ctacagggcc acaactgccc ccgccacaac 600 ccaccccgct ttcgtagttt tcatttagaa aatagagctt ttaaaaatgt cctgcctttt 660 aacgtagata taagccttcc cccactaccg taaatgtcca tttatatcat tttttatata 720 ttcttataaa aatgtaaaaa agaaaaacac cgcttctgcc ttttcactgt gttggagttt 780 tctggagtga gcactcacgc cctaagcgca cattcatgtg ggcatttctt gcgagcctcg 840 cagcctccgg aagctgtcga cttcatgaca agcattttgt gaactaggga agctcagggg 900 ggttactggc ttctcttgag tcacactgct agcaaatggc agaaccaaag ctcaaataaa 960 aataaaataa ttttcattca ttcactc 987 14 8972 DNA Homo sapiens 14 gtccaagggt agccaaggat ggctgcagct tcatatgatc agttgttaaa gcaagttgag 60 gcactgaaga tggagaactc aaatcttcga caagagctag aagataattc caatcatctt 120 acaaaactgg aaactgaggc atctaatatg aaggaagtac ttaaacaact acaaggaagt 180 attgaagatg aagctatggc ttcttctgga cagattgatt tattagagcg tcttaaagag 240 cttaacttag atagcagtaa tttccctgga gtaaaactgc ggtcaaaaat gtccctccgt 300 tcttatggaa gccgggaagg atctgtatca agccgttctg gagagtgcag tcctgttcct 360 atgggttcat ttccaagaag agggtttgta aatggaagca gagaaagtac tggatattta 420 gaagaacttg agaaagagag gtcattgctt cttgctgatc ttgacaaaga agaaaaggaa 480 aaagactggt attacgctca acttcagaat ctcactaaaa gaatagatag tcttccttta 540 actgaaaatt tttccttaca aacagatatg accagaaggc aattggaata tgaagcaagg 600 caaatcagag ttgcgatgga agaacaacta ggtacctgcc aggatatgga aaaacgagca 660 cagcgaagaa tagccagaat tcagcaaatc gaaaaggaca tacttcgtat acgacagctt 720 ttacagtccc aagcaacaga agcagagagg tcatctcaga acaagcatga aaccggctca 780 catgatgctg agcggcagaa tgaaggtcaa ggagtgggag aaatcaacat ggcaacttct 840 ggtaatggtc agggttcaac tacacgaatg gaccatgaaa cagccagtgt tttgagttct 900 agtagcacac actctgcacc tcgaaggctg acaagtcatc tgggaaccaa ggtggaaatg 960 gtgtattcat tgttgtcaat gcttggtact catgataagg atgatatgtc gcgaactttg 1020 ctagctatgt ctagctccca agacagctgt atatccatgc gacagtctgg atgtcttcct 1080 ctcctcatcc agcttttaca tggcaatgac aaagactctg tattgttggg aaattcccgg 1140 ggcagtaaag aggctcgggc cagggccagt gcagcactcc acaacatcat tcactcacag 1200 cctgatgaca agagaggcag gcgtgaaatc cgagtccttc atcttttgga acagatacgc 1260 gcttactgtg aaacctgttg ggagtggcag gaagctcatg aaccaggcat ggaccaggac 1320 aaaaatccaa tgccagctcc tgttgaacat cagatctgtc ctgctgtgtg tgttctaatg 1380 aaactttcat ttgatgaaga gcatagacat gcaatgaatg aactaggggg actacaggcc 1440 attgcagaat tattgcaagt ggactgtgaa atgtacgggc ttactaatga ccactacagt 1500 attacactaa gacgatatgc tggaatggct ttgacaaact tgacttttgg agatgtagcc 1560 aacaaggcta cgctatgctc tatgaaaggc tgcatgagag cacttgtggc ccaactaaaa 1620 tctgaaagtg aagacttaca gcaggttatt gcaagtgttt tgaggaattt gtcttggcga 1680 gcagatgtaa atagtaaaaa gacgttgcga gaagttggaa gtgtgaaagc attgatggaa 1740 tgtgctttag aagttaaaaa ggaatcaacc ctcaaaagcg tattgagtgc cttatggaat 1800 ttgtcagcac attgcactga gaataaagct gatatatgtg ctgtagatgg tgcacttgca 1860 tttttggttg gcactcttac ttaccggagc cagacaaaca ctttagccat tattgaaagt 1920 ggaggtggga tattacggaa tgtgtccagc ttgatagcta caaatgagga ccacaggcaa 1980 atcctaagag agaacaactg tctacaaact ttattacaac acttaaaatc tcatagtttg 2040 acaatagtca gtaatgcatg tggaactttg tggaatctct cagcaagaaa tcctaaagac 2100 caggaagcat tatgggacat gggggcagtt agcatgctca agaacctcat tcattcaaag 2160 cacaaaatga ttgctatggg aagtgctgca gctttaagga atctcatggc aaataggcct 2220 gcgaagtaca aggatgccaa tattatgtct cctggctcaa gcttgccatc tcttcatgtt 2280 aggaaacaaa aagccctaga agcagaatta gatgctcagc acttatcaga aacttttgac 2340 aatatagaca atttaagtcc caaggcatct catcgtagta agcagagaca caagcaaagt 2400 ctctatggtg attatgtttt tgacaccaat cgacatgatg ataataggtc agacaatttt 2460 aatactggca acatgactgt cctttcacca tatttgaata ctacagtgtt acccagctcc 2520 tcttcatcaa gaggaagctt agatagttct cgttctgaaa aagatagaag tttggagaga 2580 gaacgcggaa ttggtctagg caactaccat ccagcaacag aaaatccagg aacttcttca 2640 aagcgaggtt tgcagatctc caccactgca gcccagattg ccaaagtcat ggaagaagtg 2700 tcagccattc atacctctca ggaagacaga agttctgggt ctaccactga attacattgt 2760 gtgacagatg agagaaatgc acttagaaga agctctgctg cccatacaca ttcaaacact 2820 tacaatttca ctaagtcgga aaattcaaat aggacatgtt ctatgcctta tgccaaatta 2880 gaatacaaga gatcttcaaa tgatagttta aatagtgtca gtagtagtga tggttatggt 2940 aaaagaggtc aaatgaaacc ctcgattgaa tcctattctg aagatgatga aagtaagttt 3000 tgcagttatg gtcaataccc agccgaccta gcccataaaa tacatagtgc aaatcatatg 3060 gatgataatg atggagaact agatacacca ataaattata gtcttaaata ttcagatgag 3120 cagttgaact ctggaaggca aagtccttca cagaatgaaa gatgggcaag acccaaacac 3180 ataatagaag atgaaataaa acaaagtgag caaagacaat caaggaatca aagtacaact 3240 tatcctgttt atactgagag cactgatgat aaacacctca agttccaacc acattttgga 3300 cagcaggaat gtgtttctcc atacaggtca cggggagcca atggttcaga aacaaatcga 3360 gtgggttcta atcatggaat taatcaaaat gtaagccagt ctttgtgtca agaagatgac 3420 tatgaagatg ataagcctac caattatagt gaacgttact ctgaagaaga acagcatgaa 3480 gaagaagaga gaccaacaaa ttatagcata aaatataatg aagagaaacg tcatgtggat 3540 cagcctattg attatagttt aaaatatgcc acagatattc cttcatcaca gaaacagtca 3600 ttttcattct caaagagttc atctggacaa agcagtaaaa ccgaacatat gtcttcaagc 3660 agtgagaata cgtccacacc ttcatctaat gccaagaggc agaatcagct ccatccaagt 3720 tctgcacaga gtagaagtgg tcagcctcaa aaggctgcca cttgcaaagt ttcttctatt 3780 aaccaagaaa caatacagac ttattgtgta gaagatactc caatatgttt ttcaagatgt 3840 agttcattat catctttgtc atcagctgaa gatgaaatag gatgtaatca gacgacacag 3900 gaagcagatt ctgctaatac cctgcaaata gcagaaataa aagaaaagat tggaactagg 3960 tcagctgaag atcctgtgag cgaagttcca gcagtgtcac agcaccctag aaccaaatcc 4020 agcagactgc agggttctag tttatcttca gaatcagcca ggcacaaagc tgttgaattt 4080 tcttcaggag cgaaatctcc ctccaaaagt ggtgctcaga cacccaaaag tccacctgaa 4140 cactatgttc aggagacccc actcatgttt agcagatgta cttctgtcag ttcacttgat 4200 agttttgaga gtcgttcgat tgccagctcc gttcagagtg aaccatgcag tggaatggta 4260 agtggcatta taagccccag tgatcttcca gatagccctg gacaaaccat gccaccaagc 4320 agaagtaaaa cacctccacc acctcctcaa acagctcaaa ccaagcgaga agtacctaaa 4380 aataaagcac ctactgctga aaagagagag agtggaccta agcaagctgc agtaaatgct 4440 gcagttcaga gggtccaggt tcttccagat gctgatactt tattacattt tgccacggaa 4500 agtactccag atggattttc ttgttcatcc agcctgagtg ctctgagcct cgatgagcca 4560 tttatacaga aagatgtgga attaagaata atgcctccag ttcaggaaaa tgacaatggg 4620 aatgaaacag aatcagagca gcctaaagaa tcaaatgaaa accaagagaa agaggcagaa 4680 aaaactattg attctgaaaa ggacctatta gatgattcag atgatgatga tattgaaata 4740 ctagaagaat gtattatttc tgccatgcca acaaagtcat cacgtaaagc aaaaaagcca 4800 gcccagactg cttcaaaatt acctccacct gtggcaagga aaccaagtca gctgcctgtg 4860 tacaaacttc taccatcaca aaacaggttg caaccccaaa agcatgttag ttttacaccg 4920 ggggatgata tgccacgggt gtattgtgtt gaagggacac ctataaactt ttccacagct 4980 acatctctaa gtgatctaac aatcgaatcc cctccaaatg agttagctgc tggagaagga 5040 gttagaggag gagcacagtc aggtgaattt gaaaaacgag ataccattcc tacagaaggc 5100 agaagtacag atgaggctca aggaggaaaa acctcatctg taaccatacc tgaattggat 5160 gacaataaag cagaggaagg tgatattctt gcagaatgca ttaattctgc tatgcccaaa 5220 gggaaaagtc acaagccttt ccgtgtgaaa aagataatgg accaggtcca gcaagcatct 5280 gcgtcgtctt ctgcacccaa caaaaatcag ttagatggta agaaaaagaa accaacttca 5340 ccagtaaaac ctataccaca aaatactgaa tataggacac gtgtaagaaa aaatgcagac 5400 tcaaaaaata atttaaatgc tgagagagtt ttctcagaca acaaagattc aaagaaacag 5460 aatttgaaaa ataattccaa ggacttcaat gataagctcc caaataatga agatagagtc 5520 agaggaagtt ttgcttttga ttcacctcat cattacacgc ctattgaagg aactccttac 5580 tgtttttcac gaaatgattc tttgagttct ctagattttg atgatgatga tgttgacctt 5640 tccagggaaa aggctgaatt aagaaaggca aaagaaaata aggaatcaga ggctaaagtt 5700 accagccaca cagaactaac ctccaaccaa caatcagcta ataagacaca agctattgca 5760 aagcagccaa taaatcgagg tcagcctaaa cccatacttc agaaacaatc cacttttccc 5820 cagtcatcca aagacatacc agacagaggg gcagcaactg atgaaaagtt acagaatttt 5880 gctattgaaa atactccagt ttgcttttct cataattcct ctctgagttc tctcagtgac 5940 attgaccaag aaaacaacaa taaagaaaat gaacctatca aagagactga gccccctgac 6000 tcacagggag aaccaagtaa acctcaagca tcaggctatg ctcctaaatc atttcatgtt 6060 gaagataccc cagtttgttt ctcaagaaac agttctctca gttctcttag tattgactct 6120 gaagatgacc tgttgcagga atgtataagc tccgcaatgc caaaaaagaa aaagccttca 6180 agactcaagg gtgataatga aaaacatagt cccagaaata tgggtggcat attaggtgaa 6240 gatctgacac ttgatttgaa agatatacag agaccagatt cagaacatgg tctatcccct 6300 gattcagaaa attttgattg gaaagctatt caggaaggtg caaattccat agtaagtagt 6360 ttacatcaag ctgctgctgc tgcatgttta tctagacaag cttcgtctga ttcagattcc 6420 atcctttccc tgaaatcagg aatctctctg ggatcaccat ttcatcttac acctgatcaa 6480 gaagaaaaac cctttacaag taataaaggc ccacgaattc taaaaccagg ggagaaaagt 6540 acattggaaa ctaaaaagat agaatctgaa agtaaaggaa tcaaaggagg aaaaaaagtt 6600 tataaaagtt tgattactgg aaaagttcga tctaattcag aaatttcagg ccaaatgaaa 6660 cagccccttc aagcaaacat gccttcaatc tctcgaggca ggacaatgat tcatattcca 6720 ggagttcgaa atagctcctc aagtacaagt cctgtttcta aaaaaggccc accccttaag 6780 actccagcct ccaaaagccc tagtgaaggt caaacagcca ccacttctcc tagaggagcc 6840 aagccatctg tgaaatcaga attaagccct gttgccaggc agacatccca aataggtggg 6900 tcaagtaaag caccttctag atcaggatct agagattcga ccccttcaag acctgcccag 6960 caaccattaa gtagacctat acagtctcct ggccgaaact caatttcccc tggtagaaat 7020 ggaataagtc ctcctaacaa attatctcaa cttccaagga catcatcccc tagtactgct 7080 tcaactaagt cctcaggttc tggaaaaatg tcatatacat ctccaggtag acagatgagc 7140 caacagaacc ttaccaaaca aacaggttta tccaagaatg ccagtagtat tccaagaagt 7200 gagtctgcct ccaaaggact aaatcagatg aataatggta atggagccaa taaaaaggta 7260 gaactttcta gaatgtcttc

aactaaatca agtggaagtg aatctgatag atcagaaaga 7320 cctgtattag tacgccagtc aactttcatc aaagaagctc caagcccaac cttaagaaga 7380 aaattggagg aatctgcttc atttgaatct ctttctccat catctagacc agcttctccc 7440 actaggtccc aggcacaaac tccagtttta agtccttccc ttcctgatat gtctctatcc 7500 acacattcgt ctgttcaggc tggtggatgg cgaaaactcc cacctaatct cagtcccact 7560 atagagtata atgatggaag accagcaaag cgccatgata ttgcacggtc tcattctgaa 7620 agtccttcta gacttccaat caataggtca ggaacctgga aacgtgagca cagcaaacat 7680 tcatcatccc ttcctcgagt aagcacttgg agaagaactg gaagttcatc ttcaattctt 7740 tctgcttcat cagaatccag tgaaaaagca aaaagtgagg atgaaaaaca tgtgaactct 7800 atttcaggaa ccaaacaaag taaagaaaac caagtatccg caaaaggaac atggagaaaa 7860 ataaaagaaa atgaattttc tcccacaaat agtacttctc agaccgtttc ctcaggtgct 7920 acaaatggtg ctgaatcaaa gactctaatt tatcaaatgg cacctgctgt ttctaaaaca 7980 gaggatgttt gggtgagaat tgaggactgt cccattaaca atcctagatc tggaagatct 8040 cccacaggta atactccccc ggtgattgac agtgtttcag aaaaggcaaa tccaaacatt 8100 aaagattcaa aagataatca ggcaaaacaa aatgtgggta atggcagtgt tcccatgcgt 8160 accgtgggtt tggaaaatcg cctgaactcc tttattcagg tggatgcccc tgaccaaaaa 8220 ggaactgaga taaaaccagg acaaaataat cctgtccctg tatcagagac taatgaaagt 8280 tctatagtgg aacgtacccc attcagttct agcagctcaa gcaaacacag ttcacctagt 8340 gggactgttg ctgccagagt gactcctttt aattacaacc caagccctag gaaaagcagc 8400 gcagatagca cttcagctcg gccatctcag atcccaactc cagtgaataa caacacaaag 8460 aagcgagatt ccaaaactga cagcacagaa tccagtggaa cccaaagtcc taagcgccat 8520 tctgggtctt accttgtgac atctgtttaa aagagaggaa gaatgaaact aagaaaattc 8580 tatgttaatt acaactgcta tatagacatt ttgtttcaaa tgaaacttta aaagactgaa 8640 aaattttgta aataggtttg attcttgtta gagggttttt gttctggaag ccatatttga 8700 tagtatactt tgtcttcact ggtcttattt tgggaggcac tcttgatggt taggaaaaaa 8760 atagtaaagc caagtatgtt tgtacagtat gttttacatg tatttaaagt agcacccatc 8820 ccaacttcct ttaattattg cttgtcttaa aataatgaac actacagata gaaaatatga 8880 tatattgctg ttatcaatca tttctagatt ataaactgac taaacttaca tcagggaaaa 8940 attggtattt atgcaaaaaa aaatgttttt gt 8972 15 5711 DNA Homo sapiens 15 agctcgctga gacttcctgg accccgcacc aggctgtggg gtttctcaga taactgggcc 60 cctgcgctca ggaggccttc accctctgct ctgggtaaag ttcattggaa cagaaagaaa 120 tggatttatc tgctcttcgc gttgaagaag tacaaaatgt cattaatgct atgcagaaaa 180 tcttagagtg tcccatctgt ctggagttga tcaaggaacc tgtctccaca aagtgtgacc 240 acatattttg caaattttgc atgctgaaac ttctcaacca gaagaaaggg ccttcacagt 300 gtcctttatg taagaatgat ataaccaaaa ggagcctaca agaaagtacg agatttagtc 360 aacttgttga agagctattg aaaatcattt gtgcttttca gcttgacaca ggtttggagt 420 atgcaaacag ctataatttt gcaaaaaagg aaaataactc tcctgaacat ctaaaagatg 480 aagtttctat catccaaagt atgggctaca gaaaccgtgc caaaagactt ctacagagtg 540 aacccgaaaa tccttccttg caggaaacca gtctcagtgt ccaactctct aaccttggaa 600 ctgtgagaac tctgaggaca aagcagcgga tacaacctca aaagacgtct gtctacattg 660 aattgggatc tgattcttct gaagataccg ttaataaggc aacttattgc agtgtgggag 720 atcaagaatt gttacaaatc acccctcaag gaaccaggga tgaaatcagt ttggattctg 780 caaaaaaggc tgcttgtgaa ttttctgaga cggatgtaac aaatactgaa catcatcaac 840 ccagtaataa tgatttgaac accactgaga agcgtgcagc tgagaggcat ccagaaaagt 900 atcagggtag ttctgtttca aacttgcatg tggagccatg tggcacaaat actcatgcca 960 gctcattaca gcatgagaac agcagtttat tactcactaa agacagaatg aatgtagaaa 1020 aggctgaatt ctgtaataaa agcaaacagc ctggcttagc aaggagccaa cataacagat 1080 gggctggaag taaggaaaca tgtaatgata ggcggactcc cagcacagaa aaaaaggtag 1140 atctgaatgc tgatcccctg tgtgagagaa aagaatggaa taagcagaaa ctgccatgct 1200 cagagaatcc tagagatact gaagatgttc cttggataac actaaatagc agcattcaga 1260 aagttaatga gtggttttcc agaagtgatg aactgttagg ttctgatgac tcacatgatg 1320 gggagtctga atcaaatgcc aaagtagctg atgtattgga cgttctaaat gaggtagatg 1380 aatattctgg ttcttcagag aaaatagact tactggccag tgatcctcat gaggctttaa 1440 tatgtaaaag tgaaagagtt cactccaaat cagtagagag taatattgaa gacaaaatat 1500 ttgggaaaac ctatcggaag aaggcaagcc tccccaactt aagccatgta actgaaaatc 1560 taattatagg agcatttgtt actgagccac agataataca agagcgtccc ctcacaaata 1620 aattaaagcg taaaaggaga cctacatcag gccttcatcc tgaggatttt atcaagaaag 1680 cagatttggc agttcaaaag actcctgaaa tgataaatca gggaactaac caaacggagc 1740 agaatggtca agtgatgaat attactaata gtggtcatga gaataaaaca aaaggtgatt 1800 ctattcagaa tgagaaaaat cctaacccaa tagaatcact cgaaaaagaa tctgctttca 1860 aaacgaaagc tgaacctata agcagcagta taagcaatat ggaactcgaa ttaaatatcc 1920 acaattcaaa agcacctaaa aagaataggc tgaggaggaa gtcttctacc aggcatattc 1980 atgcgcttga actagtagtc agtagaaatc taagcccacc taattgtact gaattgcaaa 2040 ttgatagttg ttctagcagt gaagagataa agaaaaaaaa gtacaaccaa atgccagtca 2100 ggcacagcag aaacctacaa ctcatggaag gtaaagaacc tgcaactgga gccaagaaga 2160 gtaacaagcc aaatgaacag acaagtaaaa gacatgacag cgatactttc ccagagctga 2220 agttaacaaa tgcacctggt tcttttacta agtgttcaaa taccagtgaa cttaaagaat 2280 ttgtcaatcc tagccttcca agagaagaaa aagaagagaa actagaaaca gttaaagtgt 2340 ctaataatgc tgaagacccc aaagatctca tgttaagtgg agaaagggtt ttgcaaactg 2400 aaagatctgt agagagtagc agtatttcat tggtacctgg tactgattat ggcactcagg 2460 aaagtatctc gttactggaa gttagcactc tagggaaggc aaaaacagaa ccaaataaat 2520 gtgtgagtca gtgtgcagca tttgaaaacc ccaagggact aattcatggt tgttccaaag 2580 ataatagaaa tgacacagaa ggctttaagt atccattggg acatgaagtt aaccacagtc 2640 gggaaacaag catagaaatg gaagaaagtg aacttgatgc tcagtatttg cagaatacat 2700 tcaaggtttc aaagcgccag tcatttgctc cgttttcaaa tccaggaaat gcagaagagg 2760 aatgtgcaac attctctgcc cactctgggt ccttaaagaa acaaagtcca aaagtcactt 2820 ttgaatgtga acaaaaggaa gaaaatcaag gaaagaatga gtctaatatc aagcctgtac 2880 agacagttaa tatcactgca ggctttcctg tggttggtca gaaagataag ccagttgata 2940 atgccaaatg tagtatcaaa ggaggctcta ggttttgtct atcatctcag ttcagaggca 3000 acgaaactgg actcattact ccaaataaac atggactttt acaaaaccca tatcgtatac 3060 caccactttt tcccatcaag tcatttgtta aaactaaatg taagaaaaat ctgctagagg 3120 aaaactttga ggaacattca atgtcacctg aaagagaaat gggaaatgag aacattccaa 3180 gtacagtgag cacaattagc cgtaataaca ttagagaaaa tgtttttaaa gaagccagct 3240 caagcaatat taatgaagta ggttccagta ctaatgaagt gggctccagt attaatgaaa 3300 taggttccag tgatgaaaac attcaagcag aactaggtag aaacagaggg ccaaaattga 3360 atgctatgct tagattaggg gttttgcaac ctgaggtcta taaacaaagt cttcctggaa 3420 gtaattgtaa gcatcctgaa ataaaaaagc aagaatatga agaagtagtt cagactgtta 3480 atacagattt ctctccatat ctgatttcag ataacttaga acagcctatg ggaagtagtc 3540 atgcatctca ggtttgttct gagacacctg atgacctgtt agatgatggt gaaataaagg 3600 aagatactag ttttgctgaa aatgacatta aggaaagttc tgctgttttt agcaaaagcg 3660 tccagaaagg agagcttagc aggagtccta gccctttcac ccatacacat ttggctcagg 3720 gttaccgaag aggggccaag aaattagagt cctcagaaga gaacttatct agtgaggatg 3780 aagagcttcc ctgcttccaa cacttgttat ttggtaaagt aaacaatata ccttctcagt 3840 ctactaggca tagcaccgtt gctaccgagt gtctgtctaa gaacacagag gagaatttat 3900 tatcattgaa gaatagctta aatgactgca gtaaccaggt aatattggca aaggcatctc 3960 aggaacatca ccttagtgag gaaacaaaat gttctgctag cttgttttct tcacagtgca 4020 gtgaattgga agacttgact gcaaatacaa acacccagga tcctttcttg attggttctt 4080 ccaaacaaat gaggcatcag tctgaaagcc agggagttgg tctgagtgac aaggaattgg 4140 tttcagatga tgaagaaaga ggaacgggct tggaagaaaa taatcaagaa gagcaaagca 4200 tggattcaaa cttaggtgaa gcagcatctg ggtgtgagag tgaaacaagc gtctctgaag 4260 actgctcagg gctatcctct cagagtgaca ttttaaccac tcagcagagg gataccatgc 4320 aacataacct gataaagctc cagcaggaaa tggctgaact agaagctgtg ttagaacagc 4380 atgggagcca gccttctaac agctaccctt ccatcataag tgactcttct gcccttgagg 4440 acctgcgaaa tccagaacaa agcacatcag aaaaagcagt attaacttca cagaaaagta 4500 gtgaataccc tataagccag aatccagaag gcctttctgc tgacaagttt gaggtgtctg 4560 cagatagttc taccagtaaa aataaagaac caggagtgga aaggtcatcc ccttctaaat 4620 gcccatcatt agatgatagg tggtacatgc acagttgctc tgggagtctt cagaatagaa 4680 actacccatc tcaagaggag ctcattaagg ttgttgatgt ggaggagcaa cagctggaag 4740 agtctgggcc acacgatttg acggaaacat cttacttgcc aaggcaagat ctagagggaa 4800 ccccttacct ggaatctgga atcagcctct tctctgatga ccctgaatct gatccttctg 4860 aagacagagc cccagagtca gctcgtgttg gcaacatacc atcttcaacc tctgcattga 4920 aagttcccca attgaaagtt gcagaatctg cccagagtcc agctgctgct catactactg 4980 atactgctgg gtataatgca atggaagaaa gtgtgagcag ggagaagcca gaattgacag 5040 cttcaacaga aagggtcaac aaaagaatgt ccatggtggt gtctggcctg accccagaag 5100 aatttatgct cgtgtacaag tttgccagaa aacaccacat cactttaact aatctaatta 5160 ctgaagagac tactcatgtt gttatgaaaa cagatgctga gtttgtgtgt gaacggacac 5220 tgaaatattt tctaggaatt gcgggaggaa aatgggtagt tagctatttc tgggtgaccc 5280 agtctattaa agaaagaaaa atgctgaatg agcatgattt tgaagtcaga ggagatgtgg 5340 tcaatggaag aaaccaccaa ggtccaaagc gagcaagaga atcccaggac agaaagatct 5400 tcagggggct agaaatctgt tgctatgggc ccttcaccaa catgcccaca gatcaactgg 5460 aatggatggt acagctgtgt ggtgcttctg tggtgaagga gctttcatca ttcacccttg 5520 gcacaggtgt ccacccaatt gtggttgtgc agccagatgc ctggacagag gacaatggct 5580 tccatgcaat tgggcagatg tgtgaggcac ctgtggtgac ccgagagtgg gtgttggaca 5640 gtgtagcact ctaccagtgc caggagctgg acacctacct gataccccag atcccccaca 5700 gccactactg a 5711 16 2680 DNA Homo sapiens 16 ggttatcctg aatacatgtc taacaatttt ccttgcaacg ttagctgttg tttttcactg 60 tttccaaagg atcaaaattg cttcagaaat tggagacata tttgatttaa aaggaaaaac 120 ttgaacaaat ggacaatatg tctattacga atacaccaac aagtaatgat gcctgtctga 180 gcattgtgca tagtttgatg tgccatagac aaggtggaga gagtgaaaca tttgcaaaaa 240 gagcaattga aagtttggta aagaagctga aggagaaaaa agatgaattg gattctttaa 300 taacagctat aactacaaat ggagctcatc ctagtaaatg tgttaccata cagagaacat 360 tggatgggag gcttcaggtg gctggtcgga aaggatttcc tcatgtgatc tatgcccgtc 420 tctggaggtg gcctgatctt cacaaaaatg aactaaaaca tgttaaatat tgtcagtatg 480 cgtttgactt aaaatgtgat agtgtctgtg tgaatccata tcactacgaa cgagttgtat 540 cacctggaat tgatctctca ggattaacac tgcagagtaa tgctccatca agtatgatgg 600 tgaaggatga atatgtgcat gactttgagg gacagccatc gttgtccact gaaggacatt 660 caattcaaac catccagcat ccaccaagta atcgtgcatc gacagagaca tacagcaccc 720 cagctctgtt agccccatct gagtctaatg ctaccagcac tgccaacttt cccaacattc 780 ctgtggcttc cacaagtcag cctgccagta tactgggggg cagccatagt gaaggactgt 840 tgcagatagc atcagggcct cagccaggac agcagcagaa tggatttact ggtcagccag 900 ctacttacca tcataacagc actaccacct ggactggaag taggactgca ccatacacac 960 ctaatttgcc tcaccaccaa aacggccatc ttcagcacca cccgcctatg ccgccccatc 1020 ccggacatta ctggcctgtt cacaatgagc ttgcattcca gcctcccatt tccaatcatc 1080 ctgctcctga gtattggtgt tccattgctt actttgaaat ggatgttcag gtaggagaga 1140 catttaaggt tccttcaagc tgccctattg ttactgttga tggatacgtg gacccttctg 1200 gaggagatcg cttttgtttg ggtcaactct ccaatgtcca caggacagaa gccattgaga 1260 gagcaaggtt gcacataggc aaaggtgtgc agttggaatg taaaggtgaa ggtgatgttt 1320 gggtcaggtg ccttagtgac cacgcggtct ttgtacagag ttactactta gacagagaag 1380 ctgggcgtgc acctggagat gctgttcata agatctaccc aagtgcatat ataaaggtct 1440 ttgatttgcg tcagtgtcat cgacagatgc agcagcaggc ggctactgca caagctgcag 1500 cagctgccca ggcagcagcc gtggcaggaa acatccctgg cccaggatca gtaggtggaa 1560 tagctccagc tatcagtctg tcagctgctg ctggaattgg tgttgatgac cttcgtcgct 1620 tatgcatact caggatgagt tttgtgaaag gctggggacc ggattaccca agacagagca 1680 tcaaagaaac accttgctgg attgaaattc acttacaccg ggccctccag ctcctagacg 1740 aagtacttca taccatgccg attgcagacc cacaaccttt agactgaggt cttttaccgt 1800 tggggccctt aaccttatca ggatggtgga ctacaaaata caatcctgtt tataatctga 1860 agatatattt cacttttctt ctgctttatc ttttcataaa gggttgaaaa tgtgtttgct 1920 gccttgctcc tagcagacag aaactggatt aaaacaattt ttttttcctc ttcagaactt 1980 gtcaggcatg gctcagagct tgaagattag gagaaacaca ttcttattaa ttcttcacct 2040 gttatgtatg aaggaatcat tccagtgcta gaaaatttag ccctttaaaa cgtcttagag 2100 ccttttatct gcagaacatc gatatgtata tcattctaca gaataatcca gtattgctga 2160 ttttaaaggc agagaagttc tcaaagttaa ttcacctatg ttattttgtg tacaagttgt 2220 tattgttgaa catacttcaa aaataatgtg ccatgtgggt gagttaattt taccaagagt 2280 aactttactc tgtgtttaaa aatgaagtta ataatgtatt gtaatctttc atccaaaata 2340 ttttttgcaa gttatattag tgaagatggt ttcaattcag attgtcttgc aacttcagtt 2400 ttatttttgc caaggcaaaa aactcttaat ctgtgtgtat attgagaatc ccttaaaatt 2460 accagacaaa aaaatttaaa attacgtttg ttattcctag tggatgactg ttgatgaagt 2520 atacttttcc cctgttaaac agtagttgta ttcttctgta tttctaggca caaggttggt 2580 tgctaagaag cctataagag gaatttcttt tccttcattc atagggaaag gttttgtatt 2640 ttttaaaaca ctaaaagcag cgtcactcta cctaatgtct 2680 17 1095 DNA Homo sapiens 17 tccccgctct gctctgtccg gtcacaggac tttttgccct ctgttcccgg gtccctcagg 60 cggccaccca gtgggcacac tcccaggcgg cgctccggcc ccgcgctccc tccctctgcc 120 tttcattccc agctgtcaac atcctggaag ctttgaagct caggaaagaa gagaaatcca 180 ctgagaacag tctgtaaagg tccgtagtgc tatctacatc cagacggtgg aagggagaga 240 aagagaaaga aggtatccta ggaatacctg cctgcttaga ccctctataa aagctctgtg 300 catcctgcca ctgaggactc cgaagaggta gcagtcttct gaaagacttc aactgtgagg 360 acatgtcgtt cagatttggc caacatctca tcaagccctc tgtagtgttt ctcaaaacag 420 aactgtcctt cgctcttgtg aataggaaac ctgtggtacc aggacatgtc cttgtgtgcc 480 cgctgcggcc agtggagcgc ttccatgacc tgcgtcctga tgaagtggcc gatttgtttc 540 agacgaccca gagagtcggg acagtggtgg aaaaacattt ccatgggacc tctctcacct 600 tttccatgca ggatggcccc gaagccggac agactgtgaa gcacgttcac gtccatgttc 660 ttcccaggaa ggctggagac tttcacagga atgacagcat ctatgaggag ctccagaaac 720 atgacaagga ggactttcct gcctcttgga gatcagagga ggaaatggca gcagaagccg 780 cagctctgcg ggtctacttt cagtgacaca gatgtttttc agatcctgaa ttccagcaaa 840 agagctattg ccaaccagtt tgaagaccgc ccccccgcct ctccccaaga ggaactgaat 900 cagcatgaaa atgcagtttc ttcatctcac catcctgtat tcttcaacca gtgatccccc 960 acctcggtca ctccaactcc cttaaaatac ctagacctaa acggctcaga caggcagatt 1020 tgaggtttcc ccctgtctcc ttattcggca gccttatgat taaacttcct tctctgctgc 1080 aaaaaaaaaa aaaaa 1095 18 2234 DNA Homo sapiens 18 aggggacgca gcgaaaccgg ggcccgcgcc aggccagccg ggacggacgc cgatgcccgg 60 ggctgcgacg gctgcagagc gagctgccct cggaggccgg cgtggggaag atggcccagt 120 ccaccgccac ctcccctgat gggggcacca cgtttgagca cctctggagc tctctggaac 180 cagacagcac ctacttcgac cttccccagt caagccgggg gaataatgag gtggtgggcg 240 gaacggattc cagcatggac gtcttccacc tggagggcat gactacatct gtcatggccc 300 agttcaatct gctgagcagc accatggacc agatgagcag ccgcgcggcc tcggccagcc 360 cctacacccc agagcacgcc gccagcgtgc ccacccactc gccctacgca caacccagct 420 ccaccttcga caccatgtcg ccggcgcctg tcatcccctc caacaccgac taccccggac 480 cccaccactt tgaggtcact ttccagcagt ccagcacggc caagtcagcc acctggacgt 540 actccccgct cttgaagaaa ctctactgcc agatcgccaa gacatgcccc atccagatca 600 aggtgtccac cccgccaccc ccaggcactg ccatccgggc catgcctgtt tacaagaaag 660 cggagcacgt gaccgacgtc gtgaaacgct gccccaacca cgagctcggg agggacttca 720 acgaaggaca gtctgctcca gccagccacc tcatccgcgt ggaaggcaat aatctctcgc 780 agtatgtgga tgaccctgtc accggcaggc agagcgtcgt ggtgccctat gagccaccac 840 aggtggggac ggaattcacc accatcctgt acaacttcat gtgtaacagc agctgtgtag 900 ggggcatgaa ccggcggccc atcctcatca tcatcaccct ggagatgcgg gatgggcagg 960 tgctgggccg ccggtccttt gagggccgca tctgcgcctg tcctggccgc gaccgaaaag 1020 ctgatgagga ccactaccgg gagcagcagg ccctgaacga gagctccgcc aagaacgggg 1080 ccgccagcaa gcgtgccttc aagcagagcc cccctgccgt ccccgccctt ggtgccggtg 1140 tgaagaagcg gcggcatgga gacgaggaca cgtactacct tcaggtgcga ggccgggaga 1200 actttgagat cctgatgaag ctgaaagaga gcctggagct gatggagttg gtgccgcagc 1260 cactggtgga ctcctatcgg cagcagcagc agctcctaca gaggccgagt cacctacagc 1320 ccccgtccta cgggccggtc ctctcgccca tgaacaaggt gcacgggggc atgaacaagc 1380 tgccctccgt caaccagctg gtgggccagc ctcccccgca cagttcggca gctacaccca 1440 acctggggcc cgtgggcccc gggatgctca acaaccatgg ccacgcagtg ccagccaacg 1500 gcgagatgag cagcagccac agcgcccagt ccatggtctc ggggtcccac tgcactccgc 1560 caccccccta ccacgccgac cccagcctcg tcagtttttt aacaggattg gggtgtccaa 1620 actgcatcga gtatttcacc tcccaagggt tacagagcat ttaccacctg cagaacctga 1680 ccattgagga cctgggggcc ctgaagatcc ccgagcagta ccgcatgacc atctggcggg 1740 gcctgcagga cctgaagcag ggccacgact acagcaccgc gcagcagctg ctccgctcta 1800 gcaacgcggc caccatctcc atcggcggct caggggaact gcagcgccag cgggtcatgg 1860 aggccgtgca cttccgcgtg cgccacacca tcaccatccc caaccgcggc ggcccaggcg 1920 gcggccctga cgagtgggcg gacttcggct tcgacctgcc cgactgcaag gcccgcaagc 1980 agcccatcaa ggaggagttc acggaggccg agatccactg agggcctcgc ctggctgcag 2040 cctgcgccac cgcccagaga cccaagctgc ctcccctctc cttcctgtgt gtccaaaact 2100 gcctcaggag gcaggacctt cgggctgtgc ccggggaaag gcaaggtccg gcccatcccc 2160 aggcacctca caggccccag gaaaggccca gccaccgaag ccgcctgtgg acagcctgag 2220 tcacctgcag aacc 2234 19 4344 DNA Homo sapiens 19 atggcctcgg ctggtaacgc cgccgagccc caggaccgcg gcggcggcgg cagcggctgt 60 atcggtgccc cgggacggcc ggctggaggc gggaggcgca gacggacggg ggggctgcgc 120 cgtgctgccg cgccggaccg ggactatctg caccggccca gctactgcga cgccgccttc 180 gctctggagc agatttccaa ggggaaggct actggccgga aagcgccact gtggctgaga 240 gcgaagtttc agagactctt atttaaactg ggttgttaca ttcaaaaaaa ctgcggcaag 300 ttcttggttg tgggcctcct catatttggg gccttcgcgg tgggattaaa agcagcgaac 360 ctcgagacca acgtggagga gctgtgggtg gaagttggag gacgagtaag tcgtgaatta 420 aattatactc gccagaagat tggagaagag gctatgttta atcctcaact catgatacag 480 acccctaaag aagaaggtgc taatgtcctg accacagaag cgctcctaca acacctggac 540 tcggcactcc aggccagccg tgtccatgta tacatgtaca acaggcagtg gaaattggaa 600 catttgtgtt acaaatcagg agagcttatc acagaaacag gttacatgga tcagataata 660 gaatatcttt acccttgttt gattattaca cctttggact gcttctggga aggggcgaaa 720 ttacagtctg ggacagcata cctcctaggt aaacctcctt tgcggtggac aaacttcgac 780 cctttggaat tcctggaaga gttaaagaaa ataaactatc aagtggacag ctgggaggaa 840 atgctgaata aggctgaggt tggtcatggt tacatggacc gcccctgcct caatccggcc 900 gatccagact gccccgccac agcccccaac aaaaattcaa ccaaacctct tgatatggcc 960 cttgttttga atggtggatg tcatggctta tccagaaagt atatgcactg gcaggaggag 1020 ttgattgtgg gtggcacagt caagaacagc actggaaaac tcgtcagcgc ccatgccctg 1080 cagaccatgt tccagttaat gactcccaag caaatgtacg agcacttcaa ggggtacgag 1140 tatgtctcac acatcaactg gaacgaggac aaagcggcag ccatcctgga ggcctggcag 1200 aggacatatg tggaggtggt tcatcagagt gtcgcacaga actccactca aaaggtgctt 1260 tccttcacca ccacgaccct ggacgacatc ctgaaatcct tctctgacgt

cagtgtcatc 1320 cgcgtggcca gcggctactt actcatgctc gcctatgcct gtctaaccat gctgcgctgg 1380 gactgctcca agtcccaggg tgccgtgggg ctggctggcg tcctgctggt tgcactgtca 1440 gtggctgcag gactgggcct gtgctcattg atcggaattt cctttaacgc tgcaacaact 1500 caggttttgc catttctcgc tcttggtgtt ggtgtggatg atgtttttct tctggcccac 1560 gccttcagtg aaacaggaca gaataaaaga atcccttttg aggacaggac cggggagtgc 1620 ctgaagcgca caggagccag cgtggccctc acgtccatca gcaatgtcac agccttcttc 1680 atggccgcgt taatcccaat tcccgctctg cgggcgttct ccctccaggc agcggtagta 1740 gtggtgttca attttgccat ggttctgctc atttttcctg caattctcag catggattta 1800 tatcgacgcg aggacaggag actggatatt ttctgctgtt ttacaagccc ctgcgtcagc 1860 agagtgattc aggttgaacc tcaggcctac accgacacac acgacaatac ccgctacagc 1920 cccccacctc cctacagcag ccacagcttt gcccatgaaa cgcagattac catgcagtcc 1980 actgtccagc tccgcacgga gtacgacccc cacacgcacg tgtactacac caccgctgag 2040 ccgcgctccg agatctctgt gcagcccgtc accgtgacac aggacaccct cagctgccag 2100 agcccagaga gcaccagctc cacaagggac ctgctctccc agttctccga ctccagcctc 2160 cactgcctcg agcccccctg tacgaagtgg acactctcat cttttgctga gaagcactat 2220 gctcctttcc tcttgaaacc aaaagccaag gtagtggtga tcttcctttt tctgggcttg 2280 ctgggggtca gcctttatgg caccacccga gtgagagacg ggctggacct tacggacatt 2340 gtacctcggg aaaccagaga atatgacttt attgctgcac aattcaaata cttttctttc 2400 tacaacatgt atatagtcac ccagaaagca gactacccga atatccagca cttactttac 2460 gacctacaca ggagtttcag taacgtgaag tatgtcatgt tggaagaaaa caaacagctt 2520 cccaaaatgt ggctgcacta cttcagagac tggcttcagg gacttcagga tgcatttgac 2580 agtgactggg aaaccgggaa aatcatgcca aacaattaca agaatggatc agacgatgga 2640 gtccttgcct acaaactcct ggtgcaaacc ggcagccgcg ataagcccat cgacatcagc 2700 cagttgacta aacagcgtct ggtggatgca gatggcatca ttaatcccag cgctttctac 2760 atctacctga cggcttgggt cagcaacgac cccgtcgcgt atgctgcctc ccaggccaac 2820 atccggccac accgaccaga atgggtccac gacaaagccg actacatgcc tgaaacaagg 2880 ctgagaatcc cggcagcaga gcccatcgag tatgcccagt tccctttcta cctcaacggg 2940 ttgcgggaca cctcagactt tgtggaggca attgaaaaag taaggaccat ctgcagcaac 3000 tatacgagcc tggggctgtc cagttacccc aacggctacc ccttcctctt ctgggagcag 3060 tacatcggcc tccgccactg gctgctgctg ttcatcagcg tggtgttggc ctgcacattc 3120 ctcgtgtgcg ctgtcttcct tctgaacccc tggacggccg ggatcattgt gatggtcctg 3180 gcgctgatga cggtcgagct gttcggcatg atgggcctca tcggaatcaa gctcagtgcc 3240 gtgcccgtgg tcatcctgat cgcttctgtt ggcataggag tggagttcac cgttcacgtt 3300 gctttggcct ttctgacggc catcggcgac aagaaccgca gggctgtgct tgccctggag 3360 cacatgtttg cacccgtcct ggatggcgcc gtgtccactc tgctgggagt gctgatgctg 3420 gcgggatctg agttcgactt cattgtcagg tatttctttg ctgtgctggc gatcctcacc 3480 atcctcggcg ttctcaatgg gctggttttg cttcccgtgc ttttgtcttt ctttggacca 3540 tatcctgagg tgtctccagc caacggcttg aaccgcctgc ccacaccctc ccctgagcca 3600 ccccccagcg tggtccgctt cgccatgccg cccggccaca cgcacagcgg gtctgattcc 3660 tccgactcgg agtatagttc ccagacgaca gtgtcaggcc tcagcgagga gcttcggcac 3720 tacgaggccc agcagggcgc gggaggccct gcccaccaag tgatcgtgga agccacagaa 3780 aaccccgtct tcgcccactc cactgtggtc catcccgaat ccaggcatca cccaccctcg 3840 aacccgagac agcagcccca cctggactca gggtccctgc ctcccggacg gcaaggccag 3900 cagccccgca gggacccccc cagagaaggc ttgtggccac ccctctacag accgcgcaga 3960 gacgcttttg aaatttctac tgaagggcat tctggcccta gcaatagggc ccgctggggc 4020 cctcgcgggg cccgttctca caaccctcgg aacccagcgt ccactgccat gggcagctcc 4080 gtgcccggct actgccagcc catcaccact gtgacggctt ctgcctccgt gactgtcgcc 4140 gtgcacccgc cgcctgtccc tgggcctggg cggaaccccc gagggggact ctgcccaggc 4200 taccctgaga ctgaccacgg cctgtttgag gacccccacg tgcctttcca cgtccggtgt 4260 gagaggaggg attcgaaggt ggaagtcatt gagctgcagg acgtggaatg cgaggagagg 4320 ccccggggaa gcagctccaa ctga 4344 20 4740 DNA Homo sapiens 20 ttccggtttt tctcagggga cgttgaaatt atttttgtaa cgggagtcgg gagaggacgg 60 ggcgtgcccc gcgtgcgcgc gcgtcgtcct ccccggcgct cctccacagc tcgctggctc 120 ccgccgcgga aaggcgtcat gccgcccaaa accccccgaa aaacggccgc caccgccgcc 180 gctgccgccg cggaaccccc ggcaccgccg ccgccgcccc ctcctgagga ggacccagag 240 caggacagcg gcccggagga cctgcctctc gtcaggcttg agtttgaaga aacagaagaa 300 cctgatttta ctgcattatg tcagaaatta aagataccag atcatgtcag agagagagct 360 tggttaactt gggagaaagt ttcatctgtg gatggagtat tgggaggtta tattcaaaag 420 aaaaaggaac tgtggggaat ctgtatcttt attgcacgag ttgacctaga tgagatgtcg 480 ttcactttac tgagctacag aaaaacatac gaaatcagtg tccataaatt ctttaactta 540 ctaaaagaaa ttgataccag taccaaagtt gataatgcta tgtcaagact gttgaagaag 600 tatgatgtat tgtttgcact cttcagcaaa ttggaaagga catgtgaact tatatatttg 660 acacaaccca gcagttcgat atctactgaa ataaattctg cattggtgct aaaagtttct 720 tggatcacat ttttattagc taaaggggaa gtattacaaa tggaagatga tctggtgatt 780 tcatttcagt taatgctatg tgtccttgac tattttatta aactctcacc tcccatgttg 840 ctcaaagaac catataaaac agctgttata cccattaatg gttcacctcg aacacccagg 900 cgaggtcaga acaggagtgc acggatagca aaacaactag aaaatgatac aagaattatt 960 gaagttctct gtaaagaaca tgaatgtaat atagatgagg tgaaaaatgt ttatttcaaa 1020 aattttatac cttttatgaa ttctcttgga cttgtaacat ctaatggact tccagaggtt 1080 gaaaatcttt ctaaacgata cgaagaaatt tatcttaaaa ataaagatct agatcgaaga 1140 ttatttttgg atcatgataa aactcttcag actgattcta tagacagttt tgaaacacag 1200 agaacaccac gaaaaagtaa ccttgatgaa gaggtgaata taattcctcc acacactcca 1260 gttaggactg ttatgaacac tatccaacaa ttaatgatga ttttaaattc tgcaagtgat 1320 caaccttcag aaaatctgat ttcctatttt aacaactgca cagtgaatcc aaaagaaagt 1380 atactgaaaa gagtgaagga tataggatac atctttaaag agaaatttgc taaagctgtg 1440 ggacagggtt gtgtcgaaat tggatcacag cgatacaaac ttggagttcg cttgtattac 1500 cgagtaatgg aatccatgct taaatcagaa gaagaacgat tatccattca aaattttagc 1560 aaacttctga atgacaacat ttttcatatg tctttattgg cgtgcgctct tgaggttgta 1620 atggccacat atagcagaag tacatctcag aatcttgatt ctggaacaga tttgtctttc 1680 ccatggattc tgaatgtgct taatttaaaa gcctttgatt tttacaaagt gatcgaaagt 1740 tttatcaaag cagaaggcaa cttgacaaga gaaatgataa aacatttaga acgatgtgaa 1800 catcgaatca tggaatccct tgcatggctc tcagattcac ctttatttga tcttattaaa 1860 caatcaaagg accgagaagg accaactgat caccttgaat ctgcttgtcc tcttaatctt 1920 cctctccaga ataatcacac tgcagcagat atgtatcttt ctcctgtaag atctccaaag 1980 aaaaaaggtt caactacgcg tgtaaattct actgcaaatg cagagacaca agcaacctca 2040 gccttccaga cccagaagcc attgaaatct acctctcttt cactgtttta taaaaaagtg 2100 tatcggctag cctatctccg gctaaataca ctttgtgaac gccttctgtc tgagcaccca 2160 gaattagaac atatcatctg gacccttttc cagcacaccc tgcagaatga gtatgaactc 2220 atgagagaca ggcatttgga ccaaattatg atgtgttcca tgtatggcat atgcaaagtg 2280 aagaatatag accttaaatt caaaatcatt gtaacagcat acaaggatct tcctcatgct 2340 gttcaggaga cattcaaacg tgttttgatc aaagaagagg agtatgattc tattatagta 2400 ttctataact cggtcttcat gcagagactg aaaacaaata ttttgcagta tgcttccacc 2460 aggcccccta ccttgtcacc aatacctcac attcctcgaa gcccttacaa gtttcctagt 2520 tcacccttac ggattcctgg agggaacatc tatatttcac ccctgaagag tccatataaa 2580 atttcagaag gtctgccaac accaacaaaa atgactccaa gatcaagaat cttagtatca 2640 attggtgaat cattcgggac ttctgagaag ttccagaaaa taaatcagat ggtatgtaac 2700 agcgaccgtg tgctcaaaag aagtgctgaa ggaagcaacc ctcctaaacc actgaaaaaa 2760 ctacgctttg atattgaagg atcagatgaa gcagatggaa gtaaacatct cccaggagag 2820 tccaaatttc agcagaaact ggcagaaatg acttctactc gaacacgaat gcaaaagcag 2880 aaaatgaatg atagcatgga tacctcaaac aaggaagaga aatgaggatc tcaggacctt 2940 ggtggacact gtgtacacct ctggattcat tgtctctcac agatgtgact gtataacttt 3000 cccaggttct gtttatggcc acatttaata tcttcagctc tttttgtgga tataaaatgt 3060 gcagatgcaa ttgtttgggt gagtcctaag ccacttgaaa tgttagtcat tgttatttat 3120 acaagattga aaatcttgtg taaatcctgc catttaaaaa gttgtagcag attgtttcct 3180 cttccaaagt aaaattgctg tgctttatgg atagtaagaa tggccctaga gtgggagtcc 3240 tgataaccca ggcctgtctg actactttgc cttcttttgt agcatatagg tgatgtttgc 3300 tcttgttttt attaatttat atgtatattt ttttaattta acatgaacac ccttagaaaa 3360 tgtgtcctat ctatcttcca aatgcaattt gattgactgc ccattcacca aaattatcct 3420 gaactcttct gcaaaaatgg atattattag aaattagaaa aaaattacta attttacaca 3480 ttagatttta ttttactatt ggaatctgat atactgtgtg cttgttttat aaaattttgc 3540 ttttaattaa ataaaagctg gaagcaaagt ataaccatat gatactatca tactactgaa 3600 acagatttca tacctcagaa tgtaaaagaa cttactgatt attttcttca tccaacttat 3660 gtttttaaat gaggattatt gatagtactc ttggttttta taccattcag atcactgaat 3720 ttataaagta cccatctagt acttgaaaaa gtaaagtgtt ctgccagatc ttaggtatag 3780 aggaccctaa cacagtatat cccaagtgca ctttctaatg tttctgggtc ctgaagaatt 3840 aagatacaaa ttaattttac tccataaaca gactgttaat tataggagcc ttaatttttt 3900 tttcatagag atttgtctaa ttgcatctca aaattattct gccctcctta atttgggaag 3960 gtttgtgttt tctctggaat ggtacatgtc ttccatgtat cttttgaact ggcaattgtc 4020 tatttatctt ttattttttt aagtcagtat ggtctaacac tggcatgttc aaagccacat 4080 tatttctagt ccaaaattac aagtaatcaa gggtcattat gggttaggca ttaatgtttc 4140 tatctgattt tgtgcaaaag cttcaaatta aaacagctgc attagaaaaa gaggcgcttc 4200 tcccctcccc tacacctaaa ggtgtattta aactatcttg tgtgattaac ttatttagag 4260 atgctgtaac ttaaaatagg ggatatttaa ggtagcttca gctagctttt aggaaaatca 4320 ctttgtctaa ctcagaatta tttttaaaaa gaaatctggt cttgttagaa aacaaaattt 4380 tattttgtgc tcatttaagt ttcaaactta ctattttgac agttattttg ataacaatga 4440 cactagaaaa cttgactcca tttcatcatt gtttctgcat gaatatcata caaatcagtt 4500 agtttttagg tcaagggctt actatttctg ggtcttttgc tactaagttc acattagaat 4560 tagtgccaga attttaggaa cttcagagat cgtgtattga gatttcttaa ataatgcttc 4620 agatattatt gctttattgc ttttttgtat tggttaaaac tgtacattta aaattgctat 4680 gttactattt tctacaatta atagtttgtc tattttaaaa taaattagtt gttaagagtc 4740 21 4608 DNA Homo sapiens 21 atggagaata gtcttagatg tgtttgggta cccaagctgg cttttgtact cttcggagct 60 tccttgctca gcgcgcatct tcaagtaacc ggttttcaaa ttaaagcttt cacagcactg 120 cgcttcctct cagaaccttc tgatgccgtc acaatgcggg gaggaaatgt cctcctcgac 180 tgctccgcgg agtccgaccg aggagttcca gtgatcaagt ggaagaaaga tggcattcat 240 ctggccttgg gaatggatga aaggaagcag caactttcaa atgggtctct gctgatacaa 300 aacatacttc attccagaca ccacaagcca gatgagggac tttaccaatg tgaggcatct 360 ttaggagatt ctggctcaat tattagtcgg acagcaaaag ttgcagtagc aggaccactg 420 aggttccttt cacagacaga atctgtcaca gccttcatgg gagacacagt gctactcaag 480 tgtgaagtca ttggggagcc catgccaaca atccactggc agaagaacca acaagacctg 540 actccaatcc caggtgactc ccgagtggtg gtcttgccct ctggagcatt gcagatcagc 600 cgactccaac cgggggacat tggaatttac cgatgctcag ctcgaaatcc agccagctca 660 agaacaggaa atgaagcaga agtcagaatt ttatcagatc caggactgca tagacagctg 720 tattttctgc aaagaccatc caatgtagta gccattgaag gaaaagatgc tgtcctggaa 780 tgttgtgttt ctggctatcc tccaccaagt tttacctggt tacgaggcga ggaagtcatc 840 caactcaggt ctaaaaagta ttctttattg ggtggaagca acttgcttat ctccaatgtg 900 acagatgatg acagtggaat gtatacctgt gttgtcacat ataaaaatga gaatattagt 960 gcctctgcag agctcacagt cttggttccg ccatggtttt taaatcatcc ttccaacctg 1020 tatgcctatg aaagcatgga tattgagttt gaatgtacag tctctggaaa gcctgtgccc 1080 actgtgaatt ggatgaagaa tggagatgtg gtcattccta gtgattattt tcagatagtg 1140 ggaggaagca acttacggat acttggggtg gtgaagtcag atgaaggctt ttatcaatgt 1200 gtggctgaaa atgaggctgg aaatgcccag accagtgcac agctcattgt ccctaagcct 1260 gcaatcccaa gctccagtgt cctcccttcg gctcccagag atgtggtccc tgtcttggtt 1320 tccagccgat ttgtccgtct cagctggcgc ccacctgcag aagcgaaagg gaacattcaa 1380 actttcacgg tctttttctc cagagaaggt gacaacaggg aacgagcatt gaatacaaca 1440 cagcctgggt cccttcagct cactgtggga aacctgaagc cagaagccat gtacaccttt 1500 cgagttgtgg cttacaatga atggggaccg ggagagagtt ctcaacccat caaggtggcc 1560 acacagcctg agttgcaagt tccagggcca gtagaaaacc tgcaagctgt atctacctca 1620 cctacctcaa ttcttattac ctgggaaccc cctgcctatg caaacggtcc agtccaaggt 1680 tacagattgt tctgcactga ggtgtccaca ggaaaagaac agaatataga ggttgatgga 1740 ctatcttata aactggaagg cctgaaaaaa ttcaccgaat atagtcttcg attcttagct 1800 tataatcgct atggtccggg cgtctctact gatgatataa cagtggttac actttctgac 1860 gtgccaagtg ccccgcctca gaacgtctcc ctggaagtgg tcaattcaag aagtatcaaa 1920 gttagctggc tgcctcctcc atcaggaaca caaaatggat ttattaccgg ctataaaatt 1980 cgacacagaa agacgacccg caggggtgag atggaaacac tggagccaaa caacctctgg 2040 tacctattca caggactgga gaaaggaagt cagtacagtt tccaggtgtc agccatgaca 2100 gtcaatggta ctggaccacc ttccaactgg tatactgcag agactccaga gaatgatcta 2160 gatgaatctc aagttcctga tcaaccaagc tctcttcatg tgaggcccca gactaactgc 2220 atcatcatga gttggactcc tcccttgaac ccaaacatcg tggtgcgagg ttatattatc 2280 ggttatggcg ttgggagccc ttacgctgag acagtgcgtg tggacagcaa gcagcgatat 2340 tattccattg agaggttaga gtcaagttcc cattatgtaa tctccctaaa agcttttaac 2400 aatgccggag aaggagttcc tctttatgaa agtgccacca ccaggtctat aaccgatccc 2460 actgacccag ttgattatta tcctttgctt gatgatttcc ccacctcggt cccagatctc 2520 tccaccccca tgctcccacc agtaggtgta caggctgtgg ctcttaccca tgatgctgtg 2580 agggtcagct gggcagacaa ctctgtccct aagaaccaaa agacgtctga ggtgcgactt 2640 tacaccgtcc ggtggagaac cagcttttct gcaagtgcaa aatacaagtc agaagacaca 2700 acatctctaa gttacacagc aacaggcctc aaaccaaaca caatgtatga attctcggtc 2760 atggtaacaa aaaacagaag gtccagtact tggagcatga ctgcacatgc caccacgtat 2820 gaagcagccc ccacctctgc tcccaaggac tttacagtca ttactaggga agggaagcct 2880 cgtgccgtca ttgtgagttg gcagcctccc ttggaagcca atgggaaaat tactgcttac 2940 atcttatttt ataccttgga caagaacatc ccaattgatg actggattat ggaaacaatc 3000 agtggtgata ggcttactca tcaaatcatg gatctcaacc ttgatactat gtattacttt 3060 cgaattcaag cacgaaattc aaaaggagtg gggccactct ctgatcccat cctcttcagg 3120 actctgaaag tggaacaccc tgacaaaatg gctaatgacc aaggtcgtca tggagatgga 3180 ggttattggc cagttgatac taatttgatt gatagaagca ccctaaatga gccgccaatt 3240 ggacaaatgc accccccgca tggcagtgtc actcctcaga agaacagcaa cctgcttgtg 3300 atcattgtgg tcaccgttgg tgtcatcaca gtgctggtag tggtcatcgt ggctgtgatt 3360 tgcacccgac gctcttcagc ccagcagaga aagaaacggg ccacccacag tgctggcaaa 3420 aggaagggca gccagaagga cctccgaccc cctgatcttt ggatccatca tgaagaaatg 3480 gagatgaaaa atattgaaaa gccatctggc actgaccctg caggaaggga ctctcccatc 3540 caaagttgcc aagacctcac accagtcagc cacagccagt cagaaaccca actgggaagc 3600 aaaagcacct ctcattcagg tcaagacact gaggaagcag ggagctctat gtccactctg 3660 gagaggtcgc tggctgcacg ccgagccccc cgggccaagc tcatgattcc catggatgcc 3720 cagtccaaca atcctgctgt cgtgagcgcc atcccggtgc caacgctaga aagtgcccag 3780 tacccaggaa tcctcccgtc tcccacctgt ggatatcccc acccgcagtt cactctccgg 3840 cctgtgccat tcccaacact ctcagtggac cgaggtttcg gagcaggaag aagtcagtca 3900 gtgagtgaag gaccaactac ccaacaacca cctatgctgc ccccatctca gcctgagcat 3960 tctagcagcg aggaggcacc aagcagaacc atccccacag cttgtgttcg accaactcac 4020 ccactccgca gctttgctaa tcctttgcta cctccaccaa tgagtgcaat agaaccgaaa 4080 gtcccttaca caccactttt gtctcagcca gggcccactc ttcctaagac ccatgtgaaa 4140 acagcctccc ttgggttggc tggaaaagca agatcccctt tgcttcctgt gtctgtgcca 4200 acagcccctg aagtgtctga ggagagccac aaaccaacag aggattcagc caatgtgtat 4260 gaacaggatg atctgagtga acaaatggca agtttggaag gactcatgaa gcagcttaat 4320 gccatcacag gctcagcctt ttaacatgta tttctgaatg gatgaggtga attttccggg 4380 aactttgcag cataccaatt acccataaac agcacacctg tgtccaagaa ctctaaccag 4440 tgtacaggtc acccatcagg accactcagt taaggaagat cctgaagcag ttcagaagga 4500 ataagcattc cttctttcac aggcatcagg aattgtcaaa tgatgattat gagttcccta 4560 aacaaaagca aagatgcatt ttcactgcaa tgtcaaagtt tagctgct 4608 22 8959 DNA Homo sapiens 22 ccccagcctc cttgccaacg ccccctttcc ctctccccct cccgctcggc gctgaccccc 60 catccccacc cccgtgggaa cactgggagc ctgcactcca cagaccctct ccttgcctct 120 tccctcacct cagcctccgc tccccgccct cttcccggcc cagggcgccg gcccaccctt 180 ccctccgccg ccccccggcc gcggggagga catggccgcg cacaggccgg tggaatgggt 240 ccaggccgtg gtcagccgct tcgacgagca gcttccaata aaaacaggac agcagaacac 300 acataccaaa gtcagtactg agcacaacaa ggaatgtcta atcaatattt ccaaatacaa 360 gttttctttg gttataagcg gcctcactac tattttaaag aatgttaaca atatgagaat 420 atttggagaa gctgctgaaa aaaatttata tctctctcag ttgattatat tggatacact 480 ggaaaaatgt cttgctgggc aaccaaagga cacaatgaga ttagatgaaa cgatgctggt 540 caaacagttg ctgccagaaa tctgccattt tcttcacacc tgtcgtgaag gaaaccagca 600 tgcagctgaa cttcggaatt ctgcctctgg ggttttattt tctctcagct gcaacaactt 660 caatgcagtc tttagtcgca tttctaccag gttacaggaa ttaactgttt gttcagaaga 720 caatgttgat gttcatgata tagaattgtt acagtatatc aatgtggatt gtgcaaaatt 780 aaaacgactc ctgaaggaaa cagcatttaa atttaaagcc ctaaagaagg ttgcgcagtt 840 agcagttata aatagcctgg aaaaggcatt ttggaactgg gtagaaaatt atccagatga 900 atttacaaaa ctgtaccaga tcccacagac tgatatggct gaatgtgcag aaaagctatt 960 tgacttggtg gatggttttg ctgaaagcac caaacgtaaa gcagcagttt ggccactaca 1020 aatcattctc cttatcttgt gtccagaaat aatccaggat atatccaaag acgtggttga 1080 tgaaaacaac atgaataaga agttatttct ggacagtcta cgaaaagctc ttgctggcca 1140 tggaggaagt aggcagctga cagaaagtgc tgcaattgcc tgtgtcaaac tgtgtaaagc 1200 aagtacttac atcaattggg aagataactc tgtcattttc ctacttgttc agtccatggt 1260 ggttgatctt aagaacctgc tttttaatcc aagtaagcca ttctcaagag gcagtcagcc 1320 tgcagatgtg gatctaatga ttgactgcct tgtttcttgc tttcgtataa gccctcacaa 1380 caaccaacac tttaagatct gcctggctca gaattcacct tctacatttc actatgtgct 1440 ggtaaattca ctccatcgaa tcatcaccaa ttccgcattg gattggtggc ctaagattga 1500 tgctgtgtat tgtcactcgg ttgaacttcg aaatatgttt ggtgaaacac ttcataaagc 1560 agtgcaaggt tgtggagcac acccagcaat acgaatggca ccgagtctta catttaaaga 1620 aaaagtaaca agccttaaat ttaaagaaaa acctacagac ctggagacaa gaagctataa 1680 gtatcttctc ttgtccatgg tgaaactaat tcatgcagat ccaaagctct tgctttgtaa 1740 tccaagaaaa caggggcccg aaacccaagg cagtacagca gaattaatta cagggctcgt 1800 ccaactggtc cctcagtcac acatgccaga gattgctcag gaagcaatgg aggctctgct 1860 ggttcttcat cagttagata gcattgattt gtggaatcct gatgctcctg tagaaacatt 1920 ttgggagatt agctcacaaa tgctttttta catctgcaag aaattaacta gtcatcaaat 1980 gcttagtagc acagaaattc tcaagtggtt gcgggaaata ttgatctgca ggaataaatt 2040 tcttcttaaa aataagcagg cagatagaag ttcctgtcac tttctccttt tttacggggt 2100 aggatgtgat attccttcta gtggaaatac cagtcaaatg tccatggatc atgaagaatt 2160 actacgtact cctggagcct ctctccggaa gggaaaaggg aactcctcta tggatagtgc 2220 agcaggatgc agcggaaccc ccccaatttg ccgacaagcc cagaccaaac tagaagtggc 2280 cctgtacatg tttctgtgga accctgacac tgaagctgtt ctggttgcca tgtcctgttt 2340 ccgccacctc tgtgaggaag cagatatccg gtgtggggtg gatgaagtgt cagtgcataa 2400 cctcttgccc aactataaca cattcatgga gtttgcctct gtcagcaata tgatgtcaac 2460 aggaagagca gcacttcaga aaagagtgat ggcactgctg aggcgcattg agcatcccac 2520 tgcaggaaac actgaggctt

gggaagatac acatgcaaaa tgggaacaag caacaaagct 2580 aatccttaac tatccaaaag ccaaaatgga agatggccag gctgctgaaa gccttcacaa 2640 gaccattgtt aagaggcgaa tgtcccatgt gagtggagga ggatccatag atttgtctga 2700 cacagactcc ctacaggaat ggatcaacat gactggcttc ctttgtgccc ttggaggagt 2760 gtgcctccag cagagaagca attctggcct ggcaacctat agcccaccca tgggtccagt 2820 cagtgaacgt aagggttcta tgatttcagt gatgtcttca gagggaaacg cagatacacc 2880 tgtcagcaaa tttatggatc ggctgttgtc cttaatggtg tgtaaccatg agaaagtggg 2940 acttcaaata cggaccaatg ttaaggatct ggtgggtcta gaattgagtc ctgctctgta 3000 tccaatgcta tttaacaaat tgaagaatac catcagcaag ttttttgact cccaaggaca 3060 ggttttattg actgatacca atactcaatt tgtagaacaa accatagcta taatgaagaa 3120 cttgctagat aatcatactg aaggcagctc tgaacatcta gggcaagcta gcattgaaac 3180 aatgatgtta aatctggtca ggtatgttcg tgtgcttggg aatatggtcc atgcaattca 3240 aataaaaacg aaactgtgtc aattagttga agtaatgatg gcaaggagag atgacctctc 3300 attttgccaa gagatgaaat ttaggaataa gatggtagaa tacctgacag actgggttat 3360 gggaacatca aaccaagcag cagatgatga tgtaaaatgt cttacaagag atttggacca 3420 ggcaagcatg gaagcagtag tttcacttct agctggtctc cctctgcagc ctgaagaagg 3480 agatggtgtg gaattgatgg aagccaaatc acagttattt cttaaatact tcacattatt 3540 tatgaacctt ttgaatgact gcagtgaagt tgaagatgaa agtgcgcaaa caggtggcag 3600 gaaacgtggc atgtctcgga ggctggcatc actgaggcac tgtacggtcc ttgcaatgtc 3660 aaacttactc aatgccaacg tagacagtgg tctcatgcac tccataggct taggttacca 3720 caaggatctc cagacaagag ctacatttat ggaagttctg acaaaaatcc ttcaacaagg 3780 cacagaattt gacacacttg cagaaacagt attggctgat cggtttgaga gattggtgga 3840 actggtcaca atgatgggtg atcaaggaga actccctata gcgatggctc tggccaatgt 3900 ggttccttgt tctcagtggg atgaactagc tcgagttctg gttactctgt ttgattctcg 3960 gcatttactc taccaactgc tctggaacat gttttctaaa gaagtagaat tggcagactc 4020 catgcagact ctcttccgag gcaacagctt ggccagtaaa ataatgacat tctgtttcaa 4080 ggtatatggt gctacctatc tacaaaaact cctggatcct ttattacgaa ttgtgatcac 4140 atcctctgat tggcaacatg ttagctttga agtggatcct accaggttag aaccatcaga 4200 gagccttgag gaaaaccagc ggaacctcct tcagatgact gaaaagttct tccatgccat 4260 catcagttcc tcctcagaat tcccccctca acttcgaagt gtgtgccact gtttatacca 4320 ggtggttagc cagcgtttcc ctcagaacag catcggtgca gtaggaagtg ccatgttcct 4380 cagatttatc aatcctgcca ttgtctcacc gtatgaagca gggattttag ataaaaagcc 4440 accacctaga atcgaaaggg gcttgaagtt aatgtcaaag atacttcaga gtattgccaa 4500 tcatgttctc ttcacaaaag aagaacatat gcggcctttc aatgattttg tgaaaagcaa 4560 ctttgatgca gcacgcaggt ttttccttga tatagcatct gattgtccta caagtgatgc 4620 agtaaatcat agtctttcct tcataagtga cggcaatgtg cttgctttac atcgtctact 4680 ctggaacaat caggagaaaa ttgggcagta tctttccagc aacagggatc ataaagctgt 4740 tggaagacga ccttttgata agatggcaac acttcttgca tacctgggtc ctccagagca 4800 caaacctgtg gcagatacac actggtccag ccttaacctt accagttcaa agtttgagga 4860 atttatgact aggcatcagg tacatgaaaa agaagaattc aaggctttga aaacgttaag 4920 tattttctac caagctggga cttccaaagc tgggaatcct attttttatt atgttgcacg 4980 gaggttcaaa actggtcaaa tcaatggtga tttgctgata taccatgtct tactgacttt 5040 aaagccatat tatgcaaagc catatgaaat tgtagtggac cttacccata ccgggcctag 5100 caatcgcttt aaaacagact ttctctctaa gtggtttgtt gtttttcctg gctttgctta 5160 cgacaacgtc tccgcagtct atatctataa ctgtaactcc tgggtcaggg agtacaccaa 5220 gtatcatgag cggctgctga ctggcctcaa aggtagcaaa aggcttgttt tcatagactg 5280 tcctgggaaa ctggctgagc acatagagca tgaacaacag aaactacctg ctgccacctt 5340 ggctttagaa gaggacctga aggtattcca caatgctctc aagctagctc acaaagacac 5400 caaagtttct attaaagttg gttctactgc tgtccaagta acttcagcag agcgaacaaa 5460 agtcctaggg caatcagtct ttctaaatga catttattat gcttcggaaa ttgaagaaat 5520 ctgcctagta gatgagaacc agttcacctt aaccattgca aaccagggca cgccgctcac 5580 cttcatgcac caggagtgtg aagccattgt ccagtctatc attcatatcc ggacccgctg 5640 ggaactgtca cagcccgact ctatccccca acacaccaag attcggccaa aagatgtccc 5700 tgggacactg ctcaatatcg cattacttaa tttaggcagt tctgacccga gtttacggtc 5760 agctgcctat aatcttctgt gtgccttaac ttgtaccttt aatttaaaaa tcgagggcca 5820 gttactagag acatcaggtt tatgtatccc tgccaacaac accctcttta ttgtctctat 5880 tagtaagaca ctggcagcca atgagccaca cctcacgtta gaatttttgg aagagtgtat 5940 ttctggattt agcaaatcta gtattgaatt gaaacacctt tgtttggaat acatgactcc 6000 atggctgtca aatctagttc gtttttgcaa gcataatgat gatgccaaac gacaaagagt 6060 tactgctatt cttgacaagc tgataacaat gaccatcaat gaaaaacaga tgtacccatc 6120 tattcaagca aaaatatggg gaagccttgg gcagattaca gatctgcttg atgttgtact 6180 agacagtttc atcaaaacca gtgcaacagg tggcttggga tcaataaaag ctgaggtgat 6240 ggcagatact gctgtagctt tggcttctgg aaatgtgaaa ttggtttcaa gcaaggttat 6300 tggaaggatg tgcaaaataa ttgacaagac atgcttatct ccaactccta ctttagaaca 6360 acatcttatg tgggatgata ttgctatttt agcacgctac atgctgatgc tgtccttcaa 6420 caattccctt gatgtggcag ctcatcttcc ctacctcttc cacgttgtta ctttcttagt 6480 agccacaggt ccgctctccc ttagagcttc cacacatgga ctggtcatta atatcattca 6540 ctctctgtgt acttgttcac agcttcattt tagtgaagag accaagcaag ttttgagact 6600 cagtctgaca gagttctcat tacccaaatt ttacttgctg tttggcatta gcaaagtcaa 6660 gtcagctgct gtcattgcct tccgttccag ttaccgggac aggtcattct ctcctggctc 6720 ctatgagaga gagacttttg ctttgacatc cttggaaaca gtcacagaag ctttgttgga 6780 gatcatggag gcatgcatga gagatattcc aacgtgcaag tggctggacc agtggacaga 6840 actagctcaa agatttgcat tccaatataa tccatccctg caaccaagag ctcttgttgt 6900 ctttgggtgt attagcaaac gagtgtctca tgggcagata aagcagataa tccgtattct 6960 tagcaaggca cttgagagtt gcttaaaagg acctgacact tacaacagtc aagttctgat 7020 agaagctaca gtaatagcac taaccaaatt acagccactt cttaataagg actcgcctct 7080 gcacaaagcc ctcttttggg tagctgtggc tgtgctgcag cttgatgagg tcaacttgta 7140 ttcagcaggt accgcacttc ttgaacaaaa cctgcatact ttagatagtc tccgtatatt 7200 caatgacaag agtccagagg aagtatttat ggcaatccgg aatcctctgg agtggcactg 7260 caagcaaatg gatcattttg ttggactcaa tttcaactct aactttaact ttgcattggt 7320 tggacacctt ttaaaagggt acaggcatcc ttcacctgct attgttgcaa gaacagtcag 7380 aattttacat acactactaa ctctggttaa caaacacaga aattgtgaca aatttgaagt 7440 gaatacacag agcgtggcct acttagcagc tttacttaca gtgtctgaag aagttcgaag 7500 tcgctgcagc ctaaaacata gaaagtcact tcttcttact gatatttcaa tggaaaatgt 7560 tcctatggat acatatccca ttcatcatgg tgacccttcc tataggacac taaaggagac 7620 tcagccatgg tcctctccca aaggttctga aggatacctt gcagccacct atccaactgt 7680 cggccagacc agtccccgag ccaggaaatc catgagcctg gacatggggc aaccttctca 7740 ggccaacact aagaagttgc ttggaacaag gaaaagtttt gatcacttga tatcagacac 7800 aaaggctcct aaaaggcaag aaatggaatc agggatcaca acacccccca aaatgaggag 7860 agtagcagaa actgattatg aaatggaaac tcagaggatt tcctcatcac aacagcaccc 7920 acatttacgt aaagtttcag tgtctgaatc aaatgttctc ttggatgaag aagtacttac 7980 tgatccgaag atccaggcgc tgcttcttac tgttctagct acactggtaa aatataccac 8040 agatgagttt gatcaacgaa ttctttatga atacttagca gaggccagtg ttgtgtttcc 8100 caaagtcttt cctgttgtgc ataatttgtt ggactctaag atcaacaccc tgttatcatt 8160 gtgccaagat ccaaatttgt taaatccaat ccatggaatt gtgcagagtg tggtgtacca 8220 tgaagaatcc ccaccacaat accaaacatc ttacctgcaa agttttggtt ttaatggctt 8280 gtggcggttt gcaggaccgt tttcaaagca aacacaaatt ccagactatg ctgagcttat 8340 tgttaagttt cttgatgcct tgattgacac gtacctgcct ggaattgatg aagaaaccag 8400 tgaagaatcc ctcctgactc ccacatctcc ttaccctcct gcactgcaga gccagcttag 8460 tatcactgcc aaccttaacc tttctaattc catgacctca cttgcaactt cccagcattc 8520 cccaggaatc gacaaggaga acgttgaact ctcccctacc actggccact gtaacagtgg 8580 acgaactcgc cacggatccg caagccaagt gcagaagcaa agaagcgctg gcagtttcaa 8640 acgtaatagc attaagaaga tcgtgtgaag cttgcttgct ttctttttta aaatcaactt 8700 aacatgggct cttcactagt gaccccttcc ctgtccttgc cctttccccc catgttgtaa 8760 tgctgcactt cctgttttat aatgaaccca tccggtttgc catgttgcca gatgatcaac 8820 tcttcgaagc cttgcctaaa tttaatgctg ccttttcttt aacttttttt cttctacttt 8880 tggcgtgtat ctggtatatg taagtgttca gaacaactgc aaagaaagtg ggaggtcagg 8940 aaacttttaa ctgagaaat 8959 23 2257 DNA Homo sapiens 23 acggcagccg tcagggaccg tcccccaact cccctttccg ctcaggcagg gtcctcgcgg 60 cccatgctgg ccgctgggga cccgcgcagc ccagaccgtt cccgggccgg ccagccggca 120 ccatggtggc cctgaggcct gtgcagcaac tccagggggg ctaaagggct cagagtgcag 180 gccgtggggc gcgagggtcc cgggcctgag ccccgcgcca tggccggggc catcgcttcc 240 cgcatgagct tcagctctct caagaggaag caacccaaga cgttcaccgt gaggatcgtc 300 accatggacg ccgagatgga gttcaattgc gagatgaagt ggaaagggaa ggacctcttt 360 gatttggtgt gccggactct ggggctccga gaaacctggt tctttggact gcagtacaca 420 atcaaggaca cagtggcctg gctcaaaatg gacaagaagg tactggatca tgatgtttca 480 aaggaagaac cagtcacctt tcacttcttg gccaaatttt atcctgagaa tgctgaagag 540 gagctggttc aggagatcac acaacattta ttcttcttac aggtaaagaa gcagatttta 600 gatgaaaaga tctactgccc tcctgaggct tctgtgctcc tggcttctta cgccgtccag 660 gccaagtatg gtgactacga ccccagtgtt cacaagcggg gatttttggc ccaagaggaa 720 ttgcttccaa aaagggtaat aaatctgtat cagatgactc cggaaatgtg ggaggagaga 780 attactgctt ggtacgcaga gcaccgaggc cgagccaggg atgaagctga aatggaatat 840 ctgaagatag ctcaggacct ggagatgtac ggtgtgaact actttgcaat ccggaataaa 900 aagggcacag agctgctgct tggagtggat gccctggggc ttcacattta tgaccctgag 960 aacagactga cccccaagat ctccttcccg tggaatgaaa tccgaaacat ctcgtacagt 1020 gacaaggagt ttactattaa accactggat aagaaaattg atgtcttcaa gtttaactcc 1080 tcaaagcttc gtgttaataa gctgattctc cagctatgta tcgggaacca tgatctattt 1140 atgaggagaa ggaaagccga ttctttggaa gttcagcaga tgaaagccca ggccagggag 1200 gagaaggcta gaaagcagat ggagcggcag cgcctcgctc gagagaagca gatgagggag 1260 gaggctgaac gcacgaggga tgagttggag aggaggctgc tgcagatgaa agaagaagca 1320 acaatggcca acgaagcact gatgcggtct gaggagacag ctgacctgtt ggctgaaaag 1380 gcccagatca ccgaggagga ggcaaaactt ctggcccaga aggccgcaga ggctgagcag 1440 gaaatgcagc gcatcaaggc cacagcgatt cgcacggagg aggagaagcg cctgatggag 1500 cagaaggtgc tggaagccga ggtgctggca ctgaagatgg ctgaggagtc agagaggagg 1560 gccaaagagg cagatcagct gaagcaggac ctgcaggaag cacgcgaggc ggagcgaaga 1620 gccaagcaga agctcctgga gattgccacc aagcccacgt acccgcccat gaacccaatt 1680 ccagcaccgt tgcctcctga cataccaagc ttcaacctca ttggtgacag cctgtctttc 1740 gacttcaaag atactgacat gaagcggctt tccatggaga tagagaaaga aaaagtggaa 1800 tacatggaaa agagcaagca tctgcaggag cagctcaatg aactcaagac agaaatcgag 1860 gccttgaaac tgaaagagag ggagacagct ctggatattc tgcacaatga gaactccgac 1920 aggggtggca gcagcaagca caataccatt aaaaagctca ccttgcagag cgccaagtcc 1980 cgagtggcct tctttgaaga gctctagcag gtgacccagc caccccagga cctgccactt 2040 ctcctgctac cgggaccgcg ggatggacca gatatcaaga gagccatcca tagggagctg 2100 gctgggggtt tccgtgggag ctccagaact ttccccagct gagtgaagag cccagcccct 2160 cttatgtgca attgccttga actacgaccc tgtagagatt tctctcatgg cgttctagtt 2220 ctctgacctg agtctttgtt ttaagaagta tttgtct 2257 24 2969 DNA Homo sapiens 24 ccaggcagct ggggtaagga gttcaaggca gcgcccacac ccgggggctc tccgcaaccc 60 gaccgcctgt ccgctccccc acttcccgcc ctccctccca cctactcatt cacccaccca 120 cccacccaga gccgggacgg cagcccaggc gcccgggccc cgccgtctcc tcgccgcgat 180 cctggacttc ctcttgctgc aggacccggc ttccacgtgt gtcccggagc cggcgtctca 240 gcacacgctc cgctccgggc ctgggtgcct acagcagcca gagcagcagg gagtccggga 300 cccgggcggc atctgggcca agttaggcgc cgccgaggcc agcgctgaac gtctccaggg 360 ccggaggagc cgcggggcgt ccgggtctga gccgcagcaa atgggctccg acgtgcggga 420 cctgaacgcg ctgctgcccg ccgtcccctc cctgggtggc ggcggcggct gtgccctgcc 480 tgtgagcggc gcggcgcagt gggcgccggt gctggacttt gcgcccccgg gcgcttcggc 540 ttacgggtcg ttgggcggcc ccgcgccgcc accggctccg ccgccacccc cgccgccgcc 600 gcctcactcc ttcatcaaac aggagccgag ctggggcggc gcggagccgc acgaggagca 660 gtgcctgagc gccttcactg tccacttttc cggccagttc actggcacag ccggagcctg 720 tcgctacggg cccttcggtc ctcctccgcc cagccaggcg tcatccggcc aggccaggat 780 gtttcctaac gcgccctacc tgcccagctg cctcgagagc cagcccgcta ttcgcaatca 840 gggttacagc acggtcacct tcgacgggac gcccagctac ggtcacacgc cctcgcacca 900 tgcggcgcag ttccccaacc actcattcaa gcatgaggat cccatgggcc agcagggctc 960 gctgggtgag cagcagtact cggtgccgcc cccggtctat ggctgccaca cccccaccga 1020 cagctgcacc ggcagccagg ctttgctgct gaggacgccc tacagcagtg acaatttata 1080 ccaaatgaca tcccagcttg aatgcatgac ctggaatcag atgaacttag gagccacctt 1140 aaagggccac agcacagggt acgagagcga taaccacaca acgcccatcc tctgcggagc 1200 ccaatacaga atacacacgc acggtgtctt cagaggcatt caggatgtgc gacgtgtgcc 1260 tggagtagcc ccgactcttg tacggtcggc atctgagacc agtgagaaac gccccttcat 1320 gtgtgcttac ccaggctgca ataagagata ttttaagctg tcccacttac agatgcacag 1380 caggaagcac actggtgaga aaccatacca gtgtgacttc aaggactgtg aacgaaggtt 1440 ttctcgttca gaccagctca aaagacacca aaggagacat acaggtgtga aaccattcca 1500 gtgtaaaact tgtcagcgaa agttctcccg gtccgaccac ctgaagaccc acaccaggac 1560 tcatacaggt gaaaagccct tcagctgtcg gtggccaagt tgtcagaaaa agtttgcccg 1620 gtcagatgaa ttagtccgcc atcacaacat gcatcagaga aacatgacca aactccagct 1680 ggcgctttga ggggtctccc tcggggaccg ttcagtgtcc caggcagcac agtgtgtgaa 1740 ctgctttcaa gtctgactct ccactcctcc tcactaaaaa ggaaacttca gttgatcttc 1800 ttcatccaac ttccaagaca agataccggt gcttctggaa actaccaggt gtgcctggaa 1860 gagttggtct ctgccctgcc tacttttagt tgactcacag gccctggaga agcagctaac 1920 aatgtctggt tagttaaaag cccattgcca tttggtgtgg attttctact gtaagaagag 1980 ccatagctga tcatgtcccc ctgacccttc ccttcttttt ttatgctcgt tttcgctggg 2040 gatggaatta ttgtaccatt ttctatcatg gaatatttat aggccagggc atgtgtatgt 2100 gtctgctaat gtaaactttg tcatggtttc catttactaa cagcaacagc aagaaataaa 2160 tcagagagca aggcatcggg ggtgaatctt gtctaacatt cccgaggtca gccaggctgc 2220 taacctggaa agcaggatgt agttctgcca ggcaactttt aaagctcatg catttcaagc 2280 agctgaagaa aaaatcagaa ctaaccagta cctctgtata gaaatctaaa agaattttac 2340 cattcagtta attcaatgtg aacactggca cactgctctt aagaaactat gaagatctga 2400 gatttttttg tgtatgtttt tgactctttt gagtggtaat catatgtgtc tttatagatg 2460 tacatacctc cttgcacaaa tggaggggaa ttcattttca tcactgggag tgtccttagt 2520 gtataaaaac catgctggta tatggcttca agttgtaaaa atgaaagtga ctttaaaaga 2580 aaatagggga tggtccagga tctccactga taagactgtt tttaagtaac ttaaggacct 2640 ttgggtctac aagtatatgt gaaaaaaatg agacttactg ggtgaggaaa tccattgttt 2700 aaagatggtc gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgttgtgtt gtgttttgtt 2760 ttttaaggga gggaatttat tatttaccgt tgcttgaaat tactgtgtaa atatatgtct 2820 gataatgatt tgctctttga caactaaaat taggactgta taagtactag atgcatcact 2880 gggtgttgat cttacaagat attgatgata acacttaaaa ttgtaacctg catttttcac 2940 tttgctctca attaaagtct attcaaaag 2969

Claims

1. A reagent for cancer cell detection, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome.

2. A reagent for cancer diagnosis, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome.

3-5. (canceled)

6. The reagent according to claim 1, wherein the promoter capable of regulating the expression of the gene encoding a labeling protein is a cytomegalovirus promoter or hTERT promoter.

7. (canceled)

8. A cell death-inducing agent, comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a cell death-inducing cassette comprising a gene encoding a protein associated with cell death induction and a promoter capable of regulating the expression of the gene encoding the protein is integrated in E3 region of the viral genome.

9-13. (canceled)

14. The cell death-inducing agent according to claim 8, wherein the promoter capable of regulating the gene encoding the protein associated with cell death induction is a cytomegalovirus promoter or hTERT promoter.

15-19. (canceled)

20. A method of treating or preventing cancer in a subject, comprising administering to the subject a pharmaceutical composition comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome.

21. The method according to claim 20, wherein the promoter from human telomerase is hTERT promoter.

22. The method according to claim 20, wherein the labeling protein is GFP.

23. The method according to claim 20, wherein the promoter capable of regulating the expression of the gene encoding a labeling protein is a cytomegalovirus promoter or hTERT promoter.

24. The method according to claim 20, wherein the virus is an adenovirus.

25. The reagent according to claim 2, wherein the promoter capable of regulating the expression of the gene encoding a labeling protein is a cytomegalovirus promoter or hTERT promoter.

26. A method of detecting cancer cells, comprising: a) infecting cancer cells with a reagent comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome; and b) detecting the fluorescence emitted by the cancer cells.

27. The method of claim 26, wherein the promoter capable of regulating the expression of the gene encoding a labeling protein is a cytomegalovirus promoter or hTERT promoter.

28. A method of cancer diagnosis, comprising: a) infecting cancer cells with a reagent comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of the gene encoding the labeling protein is integrated in E3 region of the viral genome; and b) detecting the fluorescence emitted by the cancer cells.

29. The method of claim 28, wherein the promoter capable of regulating the expression of the gene encoding a labeling protein is a cytomegalovirus promoter or hTERT promoter.

30. A method of inducing cell death in a target cell, comprising infecting the target cell with a cell death-inducing agent comprising a recombinant virus where a replication cassette comprising a promoter from human telomerase, an E1A gene, an IRES sequence and an E1B gene in this order is integrated in E1 region of the viral genome and a cell death-inducing cassette comprising a gene encoding a protein associated with cell death induction and a promoter capable of regulating the expression of the gene encoding the protein is integrated in E3 region of the viral genome.

31. The method of claim 30, wherein the promoter capable of regulating the gene encoding the protein associated with cell death induction is a cytomegalovirus promoter or hTERT promoter.