Abstract:
Compositions and methods for the detection and treatment of cancer are provided. It has been discovered that uterine cancer cells selectively induce the survivin promoter to express a gene product. One embodiment provides a method for detecting uterine malignancy by systemic administration of a cancer-specific vector that utilizes a Survivin promoter to drive expression of a reporter gene. The expression of the reporter gene is detectable exclusively in malignant cells, for example using conventional imagining techniques.

Description:
FIELD OF THE INVENTION 
       [0001]    The invention is generally directed to molecular biology, more particularly to the detection and treatment of cancer using nucleic acid constructs. 
       REFERENCE TO THE SEQUENCE LISTING 
       [0002]    The Sequence Listing submitted as a text file named “GRU_2016_004_ST25.txt,” created on Sep. 30, 2016, and having a size of 27,881 bytes is hereby incorporated by reference pursuant to 37 C.F.R. §1.52(e)(5). 
       BACKGROUND OF THE INVENTION 
       [0003]    Benign uterine leiomyomas (fibroids) are the most common pelvic tumor in women (estimated lifetime risk of 70 percent in white women and 80 percent in black women) (Buttram, V. C., Jr. and R. C. Reiter, Fertil Steril, 36(4): 433-445 (1981); Serden, S. P. and P. G. Brooks, J Reprod Med, 36(10): 697-699 (1991); Baird, D. et al., Am J Obstet Gynecol, 188(1):100-107 (2003)). Uterine sarcoma is rare (3 to 7 per 100,000 in the United States population) with a poor prognosis (Brooks, S. E., et al., Gynecol Oncol 93(1): 204-208 (2004)). It is well recognized that cancer is an enormous global health problem. The American Cancer Society estimates that in 2008 alone there were an estimated 12.7 million new diagnoses of cancer and 7.6 million deaths caused by cancer Reynolds, P. N., et al., Nat Biotech, 19(9): 838-842 (2001)). The time at which a cancer is detected, both at initial cancer diagnosis and during tumor recurrence, is one of the most important prognostic factors that substantially affect patient outcome, because if cancer is detected early, current treatments are likely to be more effective (Etzioni, R., et al., Nat Rev Cancer, 3(4): 243-252 (2003)). 
         [0004]    Unfortunately, the majority of cancers are detected relatively late, leading to high mortality rates. These rates are expected to double by 2030 unless more effective detection strategies and treatments are developed. To stem the tremendous loss of life caused by this terrible disease, a broadly applicable tool capable of detecting cancers in their earliest stages is urgently needed. Proper differentiation between benign and malignant uterine lesions can dramatically improve the efficacy of patient treatment modalities. However, despite marvelous progress in cancer specific blood-based biomarkers, many of such biomarkers have failed clinically because of presence of limitations such as highly variable background expression from nonmalignant tissues and tumor heterogeneity. 
         [0005]    Therefore, it is an object of the invention to provide compositions and methods for the early detection of cancer, in particular uterine cancer. 
         [0006]    It is another object of the invention to provide compositions and methods for the treatment of cancer, in particular uterine cancer. 
       SUMMARY OF THE INVENTION 
       [0007]    Compositions and methods for the detection and treatment of cancer are provided. It has been discovered that uterine cancer cells selectively induce the survivin promoter to express a gene product. One embodiment provides a method for detecting uterine malignancy by systemic administration of a cancer-specific vector that utilizes a survivin promoter to drive expression of a reporter gene. The expression of the reporter gene is detectable exclusively in malignant cells, for example using conventional imagining techniques. 
         [0008]    Methods for treating uterine cancer are also provided. One method for treating uterine cancer includes administering to a subject suspected of having uterine cancer an expression vector encoding a cytotoxic agent, wherein expression of the cytotoxic agent is under the control of a survivin promoter. The vector can be administered systemically or directly into the uterus. 
         [0009]    Another embodiment provides a uterine cell containing a viral vector, wherein the viral vector contains a survivin promoter. Preferred vectors are adenoviral vectors. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0010]      FIGS. 1A-1C  are micrographs showing X-Gal staining of human SK-UT 1 cells after transfection with Ad-Lac Z reporter gene at multiplicity of infection 1 MOI ( FIG. 1A ), 3 MOI ( FIG. 1B ) and 5 MOI ( FIG. 1C ). 
           [0011]      FIG. 2  shows the results of the luciferase assay in Leiomyosarcoma tissue, primary fibroid tissue, and myometrium transfected with constructs having a Survivin promoter at 1 MOI, Survivin promoter at 5 MOI, Secretory Leukoprotease Inhibitor promoter at 1 MOI, Secretory Leukoprotease Inhibitor promoter at 5 MOI, Heparanase promoter at 1 MOI, Heparanase promoter at 5 MOI. 
           [0012]      FIG. 3  is an image of three rows of cell culture plates. The first row from top to bottom is Leiomyosarcoma, the second row is primary fibroid, and the third row is myometrium all transfected with a vector having the survivin promoter controlling expression of luciferase. MOI for each column of plates from left to right is 2, 3, 5, and 10. 
           [0013]      FIG. 4  is a line graph of total photon emission/s versus MOI for LMS (♦), 1ryF (▪) or 1ryMyoF (triangle) cell lines infected with Ad5-Survivin-luc. 
           [0014]      FIGS. 5A and 5B  are photographs of mice injected with 5×10 6  cells transfected with Ad5-Survivin-luc. Signal shows only in the transfected LMS lesions and near zero in the non-transfected as well as the begin leiomyoma case. (P&lt;0.0001) 
           [0015]      FIG. 6A  is a photograph of mice injected with 20×10 6  transfected t-1ryF cells, transfected t-LMS cells, or LMS cells. Signal shows only on transfected t-LMS cells.  FIG. 6B  is bar graph of total photon emission (TPE) per second of LMS cells, t-1ryF cells, and t-LMS cells.  FIG. 6C  is a photograph of mice injected with LMS cells, t-LMS cells, t-LMS cells, t-1ryF cells, or t-1ryF cells 
           [0016]      FIGS. 7A-7B  show images of mice with 20×10 6  cells injected intracervically. The cells are t-LMS, t-1ryF, or LMS cells.  FIG. 7A  is shows images thirty minutes post injection.  FIG. 7B  shows images one hour post injection, and  FIG. 7C  shows images post cellular implantation with the same number of cells.  FIG. 7D  is line graph of total photon emission per second versus time for from top to bottom t-LMS, t-1ryF, and LMS cells. 
           [0017]      FIG. 8  is an image of mice injected with 5×10 6  cells intracervically. The cells are t-LMS, t-LMS, LMS, t-1ryF, and t-1ryF cells from left to right. Signal shows only in the infected LMS lesions and is near zero in the non-transfected as well as benign leiomyoma case. 
           [0018]      FIG. 9  is a DNA sequence analysis of the 5′ flanking region of the human survivin gene (SEQ ID NO:1), including the ATG start codon and sequence up and downstream thereof (adapted from Li and Altieri, et al.,  Biochem. J,  344:305-311 (1999)). The amino acid sequence for the first exon of survivin is also shown (SEQ ID NO:3). Numbering is from the initiating ATG. Canonical Sp1, Sp1-like sites and CDE/CHR elements are boxed. Arrows indicate the position of the two main transcription start sites identified by primer extension and S1 nuclease protection experiments. An upward arrow indicates the first intron-exon boundary 
           [0019]      FIG. 10  is a diagram (adapted from Zhu, et al.,  Cancer Gene Therapy,  11:256-262 (2004)) of an exemplary adenoviral vector including a survivin promoter driving expression of a lunciferase reporter open reading frame and a polyA tail cloned into the deleted E1 of an adenoviral vector backbone (pAdEasy-1). In this example, E3 is also deleted from the adenoviral vector backbone. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     I. Definitions 
       [0020]    The use of the terms “a,” “an,” “the,” and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. 
         [0021]    Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. 
         [0022]    Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. +/−10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/−5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/−2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/−1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. 
         [0023]    The term “adenovirus” as used herein refers to a non-enveloped icosahedral double-stranded DNA virus having about a linear genome of about 36 kb. 
         [0024]    The term “tumor-specific promoter” as used herein refers to a promoter which is activated specifically in a tumor cell compared to a normal cell to facilitate a transcription of a gene operably linked to the promoter. 
         [0025]    The term “nucleic acid construct” or “nucleic acid cassette” as used herein refers to a nucleotide sequence constructed for insertion to an expression vector. 
         [0026]    The term “vector” as used herein refers to a vehicle for gene transfer as that term is understood by those skilled in the art, and includes viruses, plasmids, and the like. 
         [0027]    The term “operably linked” used herein refers to the arrangement of various nucleic acid molecule elements relative to each other such that the elements are functionally connected and are able to interact with each other. 
         [0028]    The term “promoter” refers to a regulatory nucleic acid sequence, typically located upstream (5′) of a gene or protein coding sequence that, in conjunction with various elements, is responsible for regulating the expression of the gene or protein coding sequence. 
         [0029]    The term “expression control sequence” refers to a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and the like. Eukaryotic cells can utilize promoters, polyadenylation signals, and enhancers. 
       II. Nucleic Acid Constructs 
       [0030]    Nucleic acid constructs for detecting or treating uterine cancer include, but are not limited to expression vectors under the control of a Survivin promoter. For detecting cancer cells, the constructs include a reporter gene. Constructs for treating cancer encode a cytotoxic agent or oncolytic virus. Preferred constructs are viral vectors. The virus can be selected from the group consisting of adenovirus, adeno-associated virus, retrovirus, lentivirus, herpes simplex virus, and reovirus. Preferably, the virus is an adenovirus, and most preferably an adenovirus derived from primates. Adenoviruses infect both non-dividing and dividing cells unlike retroviruses and replicate as episomal elements in the nucleus without integrating with host genome, thereby not disrupting host genome. Adenoviruses are also useful for gene therapy due to high efficacy, long and safe storage, and low restriction in inserting an exogenous gene. 
         [0031]    A. Constructs for Detecting Uterine Cancer 
         [0032]    Constructs for detecting uterine cancer include but are not limited to the constructs reported in Zhu, Z., et al., Cancer Gene Therapy, 11:256-262 (2004) and Houdt, W., et al., J Neurosurg, 104:583-592 (2006) both of which are incorporated by reference in their entireties. 
         [0033]    A preferred nucleic acid construct is a viral vector containing a reporter gene under the control of a survivin promoter. The reporter gene can encode an enzyme or a luminescent or fluorescent gene product. An exemplary fluorescent gene product is Green Fluorescent Protein. The reporter gene can encode luciferase which catalyzes a reaction with luciferin to produce light. 
         [0034]    1. Preferred Survivin Promoter Sequences 
         [0035]    A nucleic acid sequence for the human Survivin gene is 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                 (SEQ ID NO: 4) 
                   
               
             
          
           
               
                     1 
                 TCTAGACATG CGGATATATT CAAGCTGGGC ACAGCACAGC AGCCCCACCC CAGGCAGCTT 
                   
               
               
                   
               
               
                    61 
                 GAAATCAGAG CTGGGGTCCA AAGGGACCAC ACCCCGAGGG ACTGTGTGGG GGTCGGGGCA 
               
               
                   
               
               
                   121 
                 CACAGGCCAC TGCTTCCCCC CGTCTTTCTC AGCCATTCCT GAAGTCAGCC TCACTCTGCT 
               
               
                   
               
               
                   181 
                 TCTCAGGGAT TTCAAATGTG CAGAGACTCT GGCACTTTTG TAGAAGCCCC TTCTGGTCCT 
               
               
                   
               
               
                   241 
                 AACTTACACC TGGATGCTGT GGGGCTGCAG CTGCTGCTCG GGCTCGGGAG GATGCTGGGG 
               
               
                   
               
               
                   301 
                 GCCCGGTGCC CATGAGCTTT TGAAGCTCCT GGAACTCGGT TTTGAGGGTG TTCAGGTCCA 
               
               
                   
               
               
                   361 
                 GGTGGACACC TGGGCTGTCC TTGTCCATGC ATTTGATGAC ATTGTGTGCA GAAGTGAAAA 
               
               
                   
               
               
                   421 
                 GGAGTTAGGC CGGGCATGCT GGCTTATGCC TGTAATCCCA GCACTTTGGG AGGCTGAGGC 
               
               
                   
               
               
                   481 
                 GGGTGGATCA CGAGGTCAGG AGTTCAATAC CAGCCTGGCC AAGATGGTGA AACCCCGTCT 
               
               
                   
               
               
                   541 
                 CTACTAAAAA TACAAAAAAA TTAGCCGGGC ATGGTGGCGG GCGCATGTAA TCCCAGCTAC 
               
               
                   
               
               
                   601 
                 TGGGGGGGCT GAGGCAGAGA ATTGCTGGAA CCCAGGAGAT GGAGGTTGCA GTGAGCCAAG 
               
               
                   
               
               
                   661 
                 ATTGTGCCAC TGCACTGCAC TCCAGCCTGG CGACAGAGCA AGACTCTGTC TCAAAAAAAA 
               
               
                   
               
               
                   721 
                 AAAAAAAAAG TGAAAAGGAG TTGTTCCTTT CCTCCCTCCT GAGGGCAGGC AACTGCTGCG 
               
               
                   
               
               
                   781 
                 GTTGCCAGTG GAGGTGGTGC GTCCTTGGTC TGTGCCTGGG GGCCACCCCA GCAGAGGCCA 
               
               
                   
               
               
                   841 
                 TGGTGGTGCC AGGGCCCGGT TAGCGAGCCA ATCAGCAGGA CCCAGGGGCG ACCTGCCAAA 
               
               
                   
               
               
                   901 
                 GTCAACTGGA TTTGATAACT GCAGCGAAGT TAAGTTTCCT GATTTTGATG ATTGTGTTGT 
               
               
                   
               
               
                   961 
                 GGTTGTGTAA GAGAATGAAG TATTTCGGGG TAGTATGGTA ATGCCTTCAA CTTACAAACG 
               
               
                   
               
               
                  1021 
                 GTTCAGGTAA ACCACCCATA TACATACATA TACATGCATG TGATATATAC ACATACAGGG 
               
               
                   
               
               
                  1081 
                 ATGTGTGTGT GTTCACATAT ATGAGGGGAG AGAGACTAGG GGAGAGAAAG TAGGTTGGGG 
               
               
                   
               
               
                  1141 
                 AGAGGGAGAG AGAAAGGAAA ACAGGAGACA GAGAGAGAGC GGGGAGTAGA GAGAGGGAAG 
               
               
                   
               
               
                  1201 
                 GGGTAAGAGA GGGAGAGGAG GAGAGAAAGG GAGGAAGAAG CAGAGAGTGA ATGTTAAAGG 
               
               
                   
               
               
                  1261 
                 AAACAGGCAA AACATAAACA GAAAATCTGG GTGAAGGGTA TATGAGTATT CTTTGTACTA 
               
               
                   
               
               
                  1321 
                 TTCTTGCAAT TATCTTTTAT TTAAATTGAC ATCGGGCCGG GCGCAGTGGC TCACATCTGT 
               
               
                   
               
               
                  1381 
                 AATCCCAGCA CTTTGGGAGG CCGAGGCAGG CAGATCACTT GAGGTCAGGA GTTTGAGACC 
               
               
                   
               
               
                  1441 
                 AGCCTGGCAA ACATGGTGAA ACCCCATCTC TACTAAAAAT ACAAAAATTA GCCTGGTGTG 
               
               
                   
               
               
                  1501 
                 GTGGTGCATG CCTTTAATCT CAGCTACTCG GGAGGCTGAG GCAGGAGAAT CGCTTGAACC 
               
               
                   
               
               
                  1561 
                 CGTGGCGGGG AGGAGGTTGC AGTGAGCTGA GATCATGCCA CTGCACTCCA GCCTGGGCGA 
               
               
                   
               
               
                  1621 
                 TAGAGCGAGA CTCAGTTTCA AATAAATAAA TAAACATCAA AATAAAAAGT TACTGTATTA 
               
               
                   
               
               
                  1681 
                 AAGAATGGGG GCGGGGTGGG AGGGGTGGGG AGAGGTTGCA AAAATAAATA AATAAATAAA 
               
               
                   
               
               
                  1741 
                 TAAACCCCAA AATGAAAAAG ACAGTGGAGG CACCAGGCCT GCGTGGGGCT GGAGGGCTAA 
               
               
                   
               
               
                  1801 
                 TAAGGCCAGG CCTCTTATCT CTGGCCATAG AACCAGAGAA GTGAGTGGAT GTGATGCCCA 
               
               
                   
               
               
                  1861 
                 GCTCCAGAAG TGACTCCAGA ACACCCTGTT CCAAAGCAGA GGACACACTG ATTTTTTTTT 
               
               
                   
               
               
                  1921 
                 TAATAGGCTG CAGGACTTAC TGTTGGTGGG ACGCCCTGCT TTGCGAAGGG AAAGGAGGAG 
               
               
                   
               
               
                  1981 
                 TTTGCCCTGA GCACAGGCCC CCACCCTCCA CTGGGCTTTC CCCAGCTCCC TTGTCTTCTT 
               
               
                   
               
               
                  2041 
                 ATCACGGTAG TGGCCCAGTC CCTGGCCCCT GACTCCAGAA GGTGGCCCTC CTGGAAACCC 
               
               
                   
               
               
                  2101 
                 AGGTCGTGCA GTCAACGATG TACTCGCCGG GACAGCGATG TCTGCTGCAC TCCATCCCTC 
               
               
                   
               
               
                  2161 
                 CCCTGTTCAT TTGTCCTTCA TGCCCGTCTG GAGTAGATGC TTTTTGCAGA GGTGGCACCC 
               
               
                   
               
               
                  2221 
                 TGTAAAGCTC TCCTGTCTGA CTTTTTTTTT TTTTTTAGAC TGAGTTTTGC TCTTGTTGCC 
               
               
                   
               
               
                  2281 
                 TAGGCTGGAG TGCAATGGCA CAATCTCAGC TCACTGCACC CTCTGCCTCC CGGGTTCAAG 
               
               
                   
               
               
                  2341 
                 CGATTCTCCT GCCTCAGCCT CCCGAGTAGT TGGGATTACA GGCATGCACC ACCACGCCCA 
               
               
                   
               
               
                  2401 
                 GCTAATTTTT GTATTTTTAG TAGAGACAAG GTTTCACCGT GATGGCCAGG CTGGTCTTGA 
               
               
                   
               
               
                  2461 
                 ACTCCAGGAC TCAAGTGATG CTCCTGCCTA GGCCTCTCAA AGTGTTGGGA TTACAGGCGT 
               
               
                   
               
               
                  2521 
                 GAGCCACTGC ACCCGGCCTG CACGCGTTCT TTGAAAGCAG TCGAGGGGGC GCTAGGTGTG 
               
               
                   
               
               
                  2581 
                 GGCAGGGACG AGCTGGCGCG GCGTCGCTGG GTGCACCGCG ACCACGGGCA GAGCCACGCG 
               
               
                   
               
               
                  2641 
                 GCGGGAGGAC TACAACTCCC GGCACACCCC GCGCCGCCCC GCCTCTACTC CCAGAAGGCC 
               
               
                   
               
               
                  2701 
                 GCGGGGGGTG GACCGCCTAA GAGGGCGTGC GCTCCCGACA TGCCCCGCGG CGCGCCATTA 
               
               
                   
               
               
                  2761 
                 ACCGCCAGAT TTGAATCGCG GGACCCGTTG GCAGAGGTGG CGGCGGCGGC    ATG   GGTGCCC 
               
               
                   
               
               
                  2821 
                 CGACGTTGCC CCCTGCCTGG CAGCCCTTTC TCAAGGACCA CCGCATCTCT ACATTCAAGA 
               
               
                   
               
               
                  2881 
                 ACTGGCCCTT CTTGGAGGGC TGCGCCTGCA CCCCGGAGCG GGTGAGACTG CCCGGCCTCC 
               
               
                   
               
               
                  2941 
                 TGGGGTCCCC CACGCCCGCC TTGCCCTGTC CCTAGCGAGG CCACTGTGAC TGGGCCTCGG 
               
               
                   
               
               
                  3001 
                 GGGTACAAGC CGCCCTCCCC TCCCCGTCCT GTCCCCAGCG AGGCCACTGT GGCTGGGCCC 
               
               
                   
               
               
                  3061 
                 CTTGGGTCCA GGCCGGCCTC CCCTCCCTGC TTTGTCCCCA TCGAGGCCTT TGTGGCTGGG 
               
               
                   
               
               
                  3121 
                 CCTCGGGGTT CCGGGCTGCC ACGTCCACTC ACGAGCTGTG CTGTCCCTTG CAGATGGCCG 
               
               
                   
               
               
                  3181 
                 AGGCTGGCTT CATCCACTGC CCCACTGAGA ACGAGCCAGA CTTGGCCCAG TGTTTCTTCT 
               
               
                   
               
               
                  3241 
                 GCTTCAAGGA GCTGGAAGGC TGGGAGCCAG ATGACGACCC CATGTAAGTC TTCTCTGGCC 
               
               
                   
               
               
                  3301 
                 AGCCTCGATG GGCTTTGTTT TGAACTGAGT TGTCAAAAGA TTTGAGTTGC AAAGACACTT 
               
               
                   
               
               
                  3361 
                 AGTATGGGAG GGTTGCTTTC CACCCTCATT GCTTCTTAAA CAGCTGTTGT GAACGGATAC 
               
               
                   
               
               
                  3421 
                 CTCTCTATAT GCTGGTGCCT TGGTGATGCT TACAACCTAA TTAAATCTCA TTTGACCAAA 
               
               
                   
               
               
                  3481 
                 ATGCCTTGGG GTGGACGTAA GATGCCTGAT GCCTTTCATG TTCAACAGAA TACATCAGCA 
               
               
                   
               
               
                  3541 
                 GACCCTGTTG TTGTGAACTC CCAGGAATGT CCAAGTGCTT TTTTTGAGAT TTTTTAAAAA 
               
               
                   
               
               
                  3601 
                 ACAGTTTAAT TGAAATATAA CCTACACAGC ACAAAAATTA CCCTTTGAAA GTGTGCACTT 
               
               
                   
               
               
                  3661 
                 CACACTTTCG GAGGCTGAGG CGGGCGGATC ACCTGAGGTC AGGAGTTCAA GACCTGCCTG 
               
               
                   
               
               
                  3721 
                 GCCAACTTGG CGAAACCCCG TCTCTACTAA AAATACAAAA ATTAGCCGGG CATGGTAGCG 
               
               
                   
               
               
                  3781 
                 CACGCCCGTA ATCCCAGCTA CTCGGGAGGC TAAGGCAGGA GAATCGCTTG AACCTGGGAG 
               
               
                   
               
               
                  3841 
                 GCGGAGGTTG CAGTGAGCCG AGATTGTGCC AATGCACTCC AGCCTCGGCG ACAGAGCGAG 
               
               
                   
               
               
                  3901 
                 ACTCCGTCAT AAAAATAAAA AATTGAAAAA AAAAAAAGAA AGAAAGCATA TACTTCAGTG 
               
               
                   
               
               
                  3961 
                 TTGTTCTGGA TTTTTTTCTT CAAGATGCCT AGTTAATGAC AATGAAATTC TGTACTCGGA 
               
               
                   
               
               
                  4021 
                 TGGTATCTGT CTTTCCACAC TGTAATGCCA TATTCTTTTC TCACCTTTTT TTCTGTCGGA 
               
               
                   
               
               
                  4081 
                 TTCAGTTGCT TCCACAGCTT TAATTTTTTT CCCCTGGAGA ATCACCCCAG TTGTTTTTCT 
               
               
                   
               
               
                  4141 
                 TTTTGGCCAG AAGAGAGTAG CTGTTTTTTT TCTTAGTATG TTTGCTATGG TGGTTATACT 
               
               
                   
               
               
                  4201 
                 GCATCCCCGT AATCACTGGG AAAAGATCAG TGGTATTCTT CTTGAAAATG AATAAGTGTT 
               
               
                   
               
               
                  4261 
                 ATGATATTTT CAGATTAGAG TTACAACTGG CTGTCTTTTT GGACTTTGTG TGGCCATGTT 
               
               
                   
               
               
                  4321 
                 TTCATTGTAA TGCAGTTCTG GTAACGGTGA TAGTCAGTTA TACAGGGAGA CTCCCCTAGC 
               
               
                   
               
               
                  4381 
                 AGAAAATGAG AGTGTGAGCT AGGGGGTCCC TTGGGGAACC CGGGGCAATA ATGCCCTTCT 
               
               
                   
               
               
                  4441 
                 CTGCCCTTAA TCCTTACAGT GGGCCGGGCA CGGTGGCTTA CGCCTGTAAT ACCAGCACTT 
               
               
                   
               
               
                  4501 
                 TGGGAGGCCG AGGCGGGCGG ATCACGAGGT CAGGAGATCG AGACCATCTT GGCTAATACG 
               
               
                   
               
               
                  4561 
                 GTGAAACCCC GTCTCCACTA AAAATACAAA AAATTAGCCG GGCGTGGTGG TGGGCGCCTG 
               
               
                   
               
               
                  4621 
                 TAGTCCCAGC TACTCGGGAG GCTGAGGCAG GAGAATGGCG TGAACCCAGG AGGCGGAGCT 
               
               
                   
               
               
                  4681 
                 TGCAGTGAGC CGAGATTGCA CCACTGCACT CCAGCCTGGG CGACAGAATG AGACTCCGTC 
               
               
                   
               
               
                  4741 
                 TCAAAAAAAA AAAAAAAAGA AAAAAATCTT TACAGTGGAT TACATAACAA TTCCAGTGAA 
               
               
                   
               
               
                  4801 
                 ATGAAATTAC TTCAAACAGT TCCTTGAGAA TGTTGGAGGG ATTTGACATG TAATTCCTTT 
               
               
                   
               
               
                  4861 
                 GGACATATAC CATGTAACAC TTTTCCAACT AATTGCTAAG GAAGTCCAGA TAAAATAGAT 
               
               
                   
               
               
                  4921 
                 ACATTAGCCA CACAGATGTG GGGGGAGATG TCCACAGGGA GAGAGAAGGT GCTAAGAGGT 
               
               
                   
               
               
                  4981 
                 GCCATATGGG AATGTGGCTT GGGCAAAGCA CTGATGCCAT CAACTTCAGA CTTGACGTCT 
               
               
                   
               
               
                  5041 
                 TACTCCTGAG GCAGAGCAGG GTGTGCCTGT GGAGGGCGTG GGGAGGTGGC CCGTGGGGAG 
               
               
                   
               
               
                  5101 
                 TGGACTGCCG CTTTAATCCC TTCAGCTGCC TTTCCGCTGT TGTTTTGATT TTTCTAGAGA 
               
               
                   
               
               
                  5161 
                 GGAACATAAA AAGCATTCGT CCGGTTGCGC TTTCCTTTCT GTCAAGAAGC AGTTTGAAGA 
               
               
                   
               
               
                  5221 
                 ATTAACCCTT GGTGAATTTT TGAAACTGGA CAGAGAAAGA GCCAAGAACA AAATTGTATG 
               
               
                   
               
               
                  5281 
                 TATTGGGAAT AAGAACTGCT CAAACCCTGT TCAATGTCTT TAGCACTAAA CTACCTAGTC 
               
               
                   
               
               
                  5341 
                 CCTCAAAGGG ACTCTGTGTT TTCCTCAGGA AGCATTTTTT TTTTTTTTCT GAGATAGAGT 
               
               
                   
               
               
                  5401 
                 TTCACTCTTG TTGCCCAGGC TGGAGTGCAA TGGTGCAATC TTGGCTCACT GCAACCTCTG 
               
               
                   
               
               
                  5461 
                 CCTCTCGGGT TCAAGTGATT CTCCTGCCTC AGCCTCCCAA GTAACTGGGA TTACAGGGAA 
               
               
                   
               
               
                  5521 
                 GTGCCACCAC ACCCAGCTAA TTTTTGTATT TTTAGTAGAG ATGGGGTTTC ACCACATTGC 
               
               
                   
               
               
                  5581 
                 CCAGGCTGGT CTTGAACTCC TGACCTCGTG ATTCGCCCAC CTTGGCCTCC CAAAGTGCTG 
               
               
                   
               
               
                  5641 
                 GGATTACAGG CGTGAACCAC CACGCCTGGC TTTTTTTTTT TTGTTCTGAG ACACAGTTTC 
               
               
                   
               
               
                  5701 
                 ACTCTGTTAC CCAGGCTGGA GTAGGGTGGC CTGATCTCGG ATCACTGCAA CCTCCGCCTC 
               
               
                   
               
               
                  5761 
                 CTGGGCTCAA GTGATTTGCC TGCTTCAGCC TCCCAAGTAG CCGAGATTAC AGGCATGTGC 
               
               
                   
               
               
                  5821 
                 CACCACACCC AGGTAATTTT TGTATTTTTG GTAGAGACGA GGTTTCACCA TGTTGGCCAG 
               
               
                   
               
               
                  5881 
                 GCTGGTTTTG AACTCCTGAC CTCAGGTGAT CCACCCGCCT CAGCCTCCCA AAGTGCTGAG 
               
               
                   
               
               
                  5941 
                 ATTATAGGTG TGAGCCACCA CACCTGGCCT CAGGAAGTAT TTTTATTTTT AAATTTATTT 
               
               
                   
               
               
                  6001 
                 ATTTATTTGA GATGGAGTCT TGCTCTGTCG CCCAGGCTAG AGTGCAGCGA CGGGATCTCG 
               
               
                   
               
               
                  6061 
                 GCTCACTGCA AGCTCCGCCC CCCAGGTTCA AGCCATTCTC CTGCCTCAGC CTCCCGAGTA 
               
               
                   
               
               
                  6121 
                 GCTGGGACTA CAGGCGCCCG CCACCACACC CGGCTAATTT TTTTGTATTT TTAGTAGAGA 
               
               
                   
               
               
                  6181 
                 CGGGTTTTCA CCGTGTTAGC CAGGAGGGTC TTGATCTCCT GACCTCGTGA TCTGCCTGCC 
               
               
                   
               
               
                  6241 
                 TCGGCCTCCC AAAGTGCTGG GATTACAGGT GTGAGCCACC ACACCCGGCT ATTTTTATTT 
               
               
                   
               
               
                  6301 
                 TTTTGAGACA GGGACTCACT CTGTCACCTG GGCTGCAGTG CAGTGGTACA CCATAGCTCA 
               
               
                   
               
               
                  6361 
                 CTGCAGCCTC GAACTCCTGA GCTCAAGTGA TCCTCCCACC TCATCCTCAC AAGTAATTGG 
               
               
                   
               
               
                  6421 
                 GACTACAGGT GCACCCCACC ATGCCCACCT AATTTATTTA TTTATTTATT TATTTATTTT 
               
               
                   
               
               
                  6481 
                 CATAGAGATG AGGGTTCCCT GTGTTGTCCA GGCTGGTCTT GAACTCCTGA GCTCACGGGA 
               
               
                   
               
               
                  6541 
                 TCCTTTTGCC TGGGCCTCCC AAAGTGCTGA GATTACAGGC ATGAGCCACC GTGCCCAGCT 
               
               
                   
               
               
                  6601 
                 AGGAATCATT TTTAAAGCCC CTAGGATGTC TGTGTGATTT TAAAGCTCCT GGAGTGTGGC 
               
               
                   
               
               
                  6661 
                 CGGTATAAGT ATATACCGGT ATAAGTAAAT CCCACATTTT GTGTCAGTAT TTACTAGAAA 
               
               
                   
               
               
                  6721 
                 CTTAGTCATT TATCTGAAGT TGAAATGTAA CTGGGCTTTA TTTATTTATT TATTTATTTA 
               
               
                   
               
               
                  6781 
                 TTTATTTTTA ATTTTTTTTT TTGAGACGAG TCTCACTTTG TCACCCAGGC TGGAGTGCAG 
               
               
                   
               
               
                  6841 
                 TGGCACGATC TCGGCTCACT GCAACCTCTG CCTCCCGGGG TCAAGCGATT CTCCTGCCTT 
               
               
                   
               
               
                  6901 
                 AGCCTCCCGA GTAGCTGGGA CTACAGGCAC GCACCACCAT GCCTGGCTAA TTTTTGTATT 
               
               
                   
               
               
                  6961 
                 TTTAGTAGAC GGGGTTTCAC CATGCTGGCC AAGCTGGTCT CAAACTCCTG ACCTTGTGAT 
               
               
                   
               
               
                  7021 
                 CTGCCCGCTT TAGCCTCCCA GAGTGCTGGG ATTACAGGCA TGAGCCACCA TGCGTGGTCT 
               
               
                   
               
               
                  7081 
                 TTTTAAAATT TTTTGATTTT TTTTTTTTTT GAGACAGAGC CTTGCTCTGT CGCCCAGGCT 
               
               
                   
               
               
                  7141 
                 GGAGTGCAGT GGCACGATCT CAGCTCACTA CAAGCTCCGC CTCCCGGGTT CACGCCATTC 
               
               
                   
               
               
                  7201 
                 TTCTGCCTCA GCCTCCTGAG TAGCTGGGAC TACAGGTGCC CACCACCACG CCTGGCTAAT 
               
               
                   
               
               
                  7261 
                 TTTTTTTGGT ATTTTTATTA GAGACAAGGT TTCATCATGT TGGCCAGGCT GGTCTCAAAC 
               
               
                   
               
               
                  7321 
                 TCCTGACCTC AAGTGATCTG CCTGCCTCGG CCTCCCAAAG CGCTGAGATT ACAGGTGTGA 
               
               
                   
               
               
                  7381 
                 TCTACTGCGC CAGGCCTGGG CGTCATATAT TCTTATTTGC TAAGTCTGGC AGCCCCACAC 
               
               
                   
               
               
                  7441 
                 AGAATAAGTA CTGGGGGATT CCATATCCTT GTAGCAAAGC CCTGGGTGGA GAGTCAGGAG 
               
               
                   
               
               
                  7501 
                 ATGTTGTAGT TCTGTCTCTG CCACTTGCAG ACTTTGAGTT TAAGCCAGTC GTGCTCATGC 
               
               
                   
               
               
                  7561 
                 TTTCCTTGCT AAATAGAGGT TAGACCCCCT ATCCCATGGT TTCTCAGGTT GCTTTTCAGC 
               
               
                   
               
               
                  7621 
                 TTGAAAATTG TATTCCTTTG TAGAGATCAG CGTAAAATAA TTCTGTCCTT ATATGTGGCT 
               
               
                   
               
               
                  7681 
                 TTATTTTAAT TTGAGACAGA GTGTCACTCA GTCGCCCAGG CTGGAGTGTG GTGGTGCGAT 
               
               
                   
               
               
                  7741 
                 CTTGGCTCAC TGCGACCTCC ACCTCCCAGG TTCAAGCGAT TCTCGTGCCT CAGGCTCCCA 
               
               
                   
               
               
                  7801 
                 AGTAGCTGAG ATTATAGGTG TGTGCCACCA GGCCCAGCTA ACTTTTGTAT TTTTAGTAGA 
               
               
                   
               
               
                  7861 
                 GACAGGGTTT TGCCATGTTG GCTAAGCTGG TCTCGAACTC CTGGCCTCAA GTGATCTGCC 
               
               
                   
               
               
                  7921 
                 CGCCTTGGCA TCCCAAAGTG CTGGGATTAC AGGTGTGAAC CACCACACCT GGCCTCAATA 
               
               
                   
               
               
                  7981 
                 TAGTGGCTTT TAAGTGCTAA GGACTGAGAT TGTGTTTTGT CAGGAAGAGG CCAGTTGTGG 
               
               
                   
               
               
                  8041 
                 GTGAAGCATG CTGTGAGAGA GCTTGTCACC TGGTTGAGGT TGTGGGAGCT GCAGCGTGGG 
               
               
                   
               
               
                  8101 
                 AACTGGAAAG TGGGCTGGGG ATCATCTTTT TCCAGGTCAG GGGTCAGCCA GCTTTTCTGC 
               
               
                   
               
               
                  8161 
                 AGCGTGCCAT AGACCATCTC TTAGCCCTCG TGGGTCAGAG TCTCTGTTGC ATATTGTCTT 
               
               
                   
               
               
                  8221 
                 TTGTTGTTTT TCACAACCTT TTAGAAACAT AAAAAGCATT CTTAGCCCGT GGGCTGGACA 
               
               
                   
               
               
                  8281 
                 AAAAAAGGCC ATGACGGGCT GTATGGATTT GGCCCAGCAG GCCCTTGCTT GCCAAGCCCT 
               
               
                   
               
               
                  8341 
                 GTTTTAGACA AGGAGCAGCT TGTGTGCCTG GAACCATCAT GGGCACAGGG GAGGAGCAGA 
               
               
                   
               
               
                  8401 
                 GTGGATGTGG AGGTGTGAGC TGGAAACCAG GTCCCAGAGC GCTGAGAAAG ACAGAGGGTT 
               
               
                   
               
               
                  8461 
                 TTTGCCCTTG CAAGTAGAGC AACTGAAATC TGACACCATC CAGTTCCAGA AAGCCCTGAA 
               
               
                   
               
               
                  8521 
                 GTGCTGGTGG ACGCTGCGGG GTGCTCCGCT CTAGGGTTAC AGGGATGAAG ATGCAGTCTG 
               
               
                   
               
               
                  8581 
                 GTAGGGGGAG TCCACTCACC TGTTGGAAGA TGTGATTAAG AAAAGTAGAC TTTCAGGGCC 
               
               
                   
               
               
                  8641 
                 GGGCATGGTG GCTCACGCCT GTAATCCCAG CACTTTGGGA GGCCGAGGCG GGTGGATCAC 
               
               
                   
               
               
                  8701 
                 GAGGTCAGGA GATCGAGACC ATCCTGGCTA ACATGGTGAA ACCCCGTCTT TACTAAAAAT 
               
               
                   
               
               
                  8761 
                 ACAAAAAATT AGCTGGGCGT GGTGGCGGGC GCCTGTAGTC CCAGCTACTC GGGAGGCTGA 
               
               
                   
               
               
                  8821 
                 GGCAGGAGAA TGGCGTGAAC CTGGGAGGTG GAGCTTGCTG TGAGCCGAGA TCGCGCCACT 
               
               
                   
               
               
                  8881 
                 GCACTCCAGC CTGGGCGACA GAGCGAGACT CCGTCTCAAA AAAAAAAAAA AAAGTAGGCT 
               
               
                   
               
               
                  8941 
                 TTCATGATGT GTGAGCTGAA GGCGCAGTAG GCAGAAGTAG AGGCCTCAGT CCCTGCAGGA 
               
               
                   
               
               
                  9001 
                 GACCCCTCGG TCTCTATCTC CTGATAGTCA GACCCAGCCA CACTGGAAAG AGGGGAGACA 
               
               
                   
               
               
                  9061 
                 TTACAGCCTG CGAGAAAAGT AGGGAGATTT AAAAACTGCT TGGCTTTTAT TTTGAACTGT 
               
               
                   
               
               
                  9121 
                 TTTTTTTGTT TGTTTGTTTT CCCCAATTCA GAATACAGAA TACTTTTATG GATTTGTTTT 
               
               
                   
               
               
                  9181 
                 TATTACTTTA ATTTTGAAAC AATATAATCT TTTTTTTGTT GTTTTTTTGA GACAGGGTCT 
               
               
                   
               
               
                  9241 
                 TACTCTGTCA CCCAGGCTGA GTGCAGTGGT GTGATCTTGG CTCACCTCAG CCTCGACCCC 
               
               
                   
               
               
                  9301 
                 CTGGGCTCAA ATGATTCTCC CACCTCAGCT TCCCAAGTAG CTGGGACCAC AGGTGCGTGT 
               
               
                   
               
               
                  9361 
                 GTTGCGCTAT ACAAATCCTG AAGACAAGGA TGCTGTTGCT GGTGATGCTG GGGATTCCCA 
               
               
                   
               
               
                  9421 
                 AGATCCCAGA TTTGATGGCA GGATGCCCCT GTCTGCTGCC TTGCCAGGGT GCCAGGAGGG 
               
               
                   
               
               
                  9481 
                 CGCTGCTGTG GAAGCTGAGG CCCGGCCATC CAGGGCGATG CATTGGGCGC TGATTCTTGT 
               
               
                   
               
               
                  9541 
                 TCCTGCTGCT GCCTCGGTGC TTAGCTTTTG AAACAATGAA ATAAATTAGA ACCAGTGTGA 
               
               
                   
               
               
                  9601 
                 AAATCGATCA GGGAATAAAT TTAATGTGGA AATAAACTGA ACAACTTAGT TCTTCATAAG 
               
               
                   
               
               
                  9661 
                 AGTTTACTTG GTAAATACTT GTGATGAGGA CAAAACGAAG CACTAGAAGG AGAGGCGAGT 
               
               
                   
               
               
                  9721 
                 TGTAGACCTG GGTGGCAGGA GTGTTTTGTT TGTTTTCTTT GGCAGGGTCT TGCTCTGTTG 
               
               
                   
               
               
                  9781 
                 CTCAGGCTGG AGTACAGTGG CACAATCACA GCTCACTATA GCCTCGACCT CCTGGACTCA 
               
               
                   
               
               
                  9841 
                 AGCAATCCTC CTGCCTCAGC CTCCCAGTAG CTGGGACTAC AGGCGCATGC CACCATGCCT 
               
               
                   
               
               
                  9901 
                 GGCTAATTTT AAATTTTTTT TTTTCTCTTT TTTGAGATGG AATCTCACTC TGTCGCCCAG 
               
               
                   
               
               
                  9961 
                 GCTGGAGTGC AGTGGCGTGA TCTCGGCTGA CGGCAAGCTC CGCCTCCCAG GTTCACTCCA 
               
               
                   
               
               
                 10021 
                 TTCGCCTGCC TCAGCCTCCC AAGTAGCTGG GACTACAGGC GCTGGGATTA CAAACCCAAA 
               
               
                   
               
               
                 10081 
                 CCCAAAGTGC TGGGATTACA GGCGTGAGCC ACTGCACCCG GCCTGTTTTG TCTTTCAATA 
               
               
                   
               
               
                 10141 
                 GCAAGAGTTG TGTTTGCTTC GCCCCTACCT TTAGTGGAAA AATGTATAAA ATGGAGATAT 
               
               
                   
               
               
                 10201 
                 TGACCTCCAC ATTGGGGTGG TTAAATTATA GCATGTATGC AAAGGAGCTT CGCTAATTTA 
               
               
                   
               
               
                 10261 
                 AGGCTTTTTT GAAAGAGAAG AAACTGAATA ATCCATGTGT GTATATATAT TTTAAAAGCC 
               
               
                   
               
               
                 10321 
                 ATGGTCATCT TTCCATATCA GTAAAGCTGA GGCTCCCTGG GACTGCAGAG TTGTCCATCA 
               
               
                   
               
               
                 10381 
                 CAGTCCATTA TAAGTGCGCT GCTGGGCCAG GTGCAGTGGC TTGTGCCTGA ATCCCAGCAC 
               
               
                   
               
               
                 10441 
                 TTTGGGAGGC CAAGGCAGGA GGATTCATTG AGCCCAGGAG TTTTGAGGCG AGCCTGGGCA 
               
               
                   
               
               
                 10501 
                 ATGTGGCCAG ACCTCATCTC TTCAAAAAAT ACACAAAAAA TTAGCCAGGC ATGGTGGCAC 
               
               
                   
               
               
                 10561 
                 GTGCCTGTAG TCTCAGCTAC TCAGGAGGCT GAGGTGGGAG GATCACTTTG AGCCTTGCAG 
               
               
                   
               
               
                 10621 
                 GTCAAAGCTG CAGTAAGCCA TGATCTTGCC ACTGCATTCC AGCCTGGATG ACAGAGCGAG 
               
               
                   
               
               
                 10681 
                 ACCCTGTCTC TAAAAAAAAA AAAAACCAAA CGGTGCACTG TTTTCTTTTT TCTTATCAAT 
               
               
                   
               
               
                 10741 
                 TTATTATTTT TAAATTAAAT TTTCTTTTAA TAATTTATAA ATTATAAATT TATATTAAAA 
               
               
                   
               
               
                 10801 
                 AATGACAAAT TTTTATTACT TATACATGAG GTAAAACTTA GGATATATAA AGTACATATT 
               
               
                   
               
               
                 10861 
                 GAAAAGTAAT TTTTTGGCTG GCACAGTGGC TCACACCTGT AATCCCAGCA CTTTGGGAGG 
               
               
                   
               
               
                 10921 
                 CCGTGGCGGG CAGATCACAT GAGATCATGA GTTCGAGACC AACCTGACCA ACATGGAGAG 
               
               
                   
               
               
                 10981 
                 ACCCCATCTC TACTAAAAAT ACAAAATTAG CCGGGGTGGT GGCGCATGCC TGTAATCCCA 
               
               
                   
               
               
                 11041 
                 GCTACTCGGG AGGCTGAGGC AGGAGAATCT CTTGAACCCG GGAGGCAGAG GTTGCGGTGA 
               
               
                   
               
               
                 11101 
                 GCCAAGATCG TGCCTTTGCA CACCAGCCTA GGCAACAAGA GCGAAAGTCC GTCTCAAAAA 
               
               
                   
               
               
                 11161 
                 AAAAGTAATT TTTTTTAAGT TAACCTCTGT CAGCAAACAA ATTTAACCCA ATAAAGGTCT 
               
               
                   
               
               
                 11221 
                 TTGTTTTTTA ATGTAGTAGA GGAGTTAGGG TTTATAAAAA ATATGGTAGG GAAGGGGGTC 
               
               
                   
               
               
                 11281 
                 CCTGGATTTG CTAATGTGAT TGTCATTTGC CCCTTAGGAG AGAGCTCTGT TAGCAGAATG 
               
               
                   
               
               
                 11341 
                 AAAAAATTGG AAGCCAGATT CAGGGAGGGA CTGGAAGCAA AAGAATTTCT GTTCGAGGAA 
               
               
                   
               
               
                 11401 
                 GAGCCTGATG TTTGCCAGGG TCTGTTTAAC TGGACATGAA GAGGAAGGCT CTGGACTTTC 
               
               
                   
               
               
                 11461 
                 CTCCAGGAGT TTCAGGAGAA AGGTAGGGCA GTGGTTAAGA GCAGAGCTCT GCCTAGACTA 
               
               
                   
               
               
                 11521 
                 GCTGGGGTGC CTAGACTAGC TGGGGTGCCC AGACTAGCTG GGGTGCCTAG ACTAGCTGGG 
               
               
                   
               
               
                 11581 
                 TACTTTGAGT GGCTCCTTCA GCCTGGACCT CGGTTTCCTC ACCTGTATAG TAGAGATATG 
               
               
                   
               
               
                 11641 
                 GGAGCACCCA GCGCAGGATC ACTGTGAACA TAAATCAGTT AATGGAGGAA GCAGGTAGAG 
               
               
                   
               
               
                 11701 
                 TGGTGCTGGG TGCATACCAA GCACTCCGTC AGTGTTTCCT GTTATTCGAT GATTAGGAGG 
               
               
                   
               
               
                 11761 
                 CAGCTTAAAC TAGAGGGAGT TGAGCTGAAT CAGGATGTTT GTCCCAGGTA GCTGGGAATC 
               
               
                   
               
               
                 11821 
                 TGCCTAGCCC AGTGCCCAGT TTATTTAGGT GCTCTCTCAG TGTTCCCTGA TTGTTTTTTC 
               
               
                   
               
               
                 11881 
                 CTTTGTCATC TTATCTACAG GATGTGACTG GGAAGCTCTG GTTTCAGTGT CATGTGTCTA 
               
               
                   
               
               
                 11941 
                 TTCTTTATTT CCAGGCAAAG GAAACCAACA ATAAGAAGAA AGAATTTGAG GAAACTGCGA 
               
               
                   
               
               
                 12001 
                 AGAAAGTGCG CCGTGCCATC GAGCAGCTGG CTGCCATGGA TTGAGGCCTC TGGCCGGAGC 
               
               
                   
               
               
                 12061 
                 TGCCTGGTCC CAGAGTGGCT GCACCACTTC CAGGGTTTAT TCCCTGGTGC CACCAGCCTT 
               
               
                   
               
               
                 12121 
                 CCTGTGGGCC CCTTAGCAAT GTCTTAGGAA AGGAGATCAA CATTTTCAAA TTAGATGTTT 
               
               
                   
               
               
                 12181 
                 CAACTGTGCT CCTGTTTTGT CTTGAAAGTG GCACCAGAGG TGCTTCTGCC TGTGCAGCGG 
               
               
                   
               
               
                 12241 
                 GTGCTGCTGG TAACAGTGGC TGCTTCTCTC TCTCTCTCTC TTTTTTGGGG GCTCATTTTT 
               
               
                   
               
               
                 12301 
                 GCTGTTTTGA TTCCCGGGCT TACCAGGTGA GAAGTGAGGG AGGAAGAAGG CAGTGTCCCT 
               
               
                   
               
               
                 12361 
                 TTTGCTAGAG CTGACAGCTT TGTTCGCGTG GGCAGAGCCT TCCACAGTGA ATGTGTCTGG 
               
               
                   
               
               
                 12421 
                 ACCTCATGTT GTTGAGGCTG TCACAGTCCT GAGTGTGGAC TTGGCAGGTG CCTGTTGAAT 
               
               
                   
               
               
                 12481 
                 CTGAGCTGCA GGTTCCTTAT CTGTCACACC TGTGCCTCCT CAGAGGACAG TTTTTTTGTT 
               
               
                   
               
               
                 12541 
                 GTTGTGTTTT TTTGTTTTTT TTTTTTGGTA GATGCATGAC TTGTGTGTGA TGAGAGAATG 
               
               
                   
               
               
                 12601 
                 GAGACAGAGT CCCTGGCTCC TCTACTGTTT AACAACATGG CTTTCTTATT TTGTTTGAAT 
               
               
                   
               
               
                 12661 
                 TGTTAATTCA CAGAATAGCA CAAACTACAA TTAAAACTAA GCACAAAGCC ATTCTAAGTC 
               
               
                   
               
               
                 12721 
                 ATTGGGGAAA CGGGGTGAAC TTCAGGTGGA TGAGGAGACA GAATAGAGTG ATAGGAAGCG 
               
               
                   
               
               
                 12781 
                 TCTGGCAGAT ACTCCTTTTG CCACTGCTGT GTGATTAGAC AGGCCCAGTG AGCCGCGGGG 
               
               
                   
               
               
                 12841 
                 CACATGCTGG CCGCTCCTCC CTCAGAAAAA GGCAGTGGCC TAAATCCTTT TTAAATGACT 
               
               
                   
               
               
                 12901 
                 TGGCTCGATG CTGTGGGGGA CTGGCTGGGC TGCTGCAGGC CGTGTGTCTG TCAGCCCAAC 
               
               
                   
               
               
                 12961 
                 CTTCACATCT GTCACGTTCT CCACACGGGG GAGAGACGCA GTCCGCCCAG GTCCCCGCTT 
               
               
                   
               
               
                 13021 
                 TCTTTGGAGG CAGCAGCTCC CGCAGGGCTG AAGTCTGGCG TAAGATGATG GATTTGATTC 
               
               
                   
               
               
                 13081 
                 GCCCTCCTCC CTGTCATAGA GCTGCAGGGT GGATTGTTAC AGCTTCGCTG GAAACCTCTG 
               
               
                   
               
               
                 13141 
                 GAGGTCATCT CGGCTGTTCC TGAGAAATAA AAAGCCTGTC ATTTCAAACA CTGCTGTGGA 
               
               
                   
               
               
                 13201 
                 CCCTACTGGG TTTTTAAAAT ATTGTCAGTT TTTCATCGTC GTCCCTAGCC TGCCAACAGC 
               
               
                   
               
               
                 13261 
                 CATCTGCCCA GACAGCCGCA GTGAGGATGA GCGTCCTGGC AGAGACGCAG TTGTCTCTGG 
               
               
                   
               
               
                 13321 
                 GCGCTTGCCA GAGCCACGAA CCCCAGACCT GTTTGTATCA TCCGGGCTCC TTCCGGGCAG 
               
               
                   
               
               
                 13381 
                 AAACAACTGA AAATGCACTT CAGACCCACT TATTTATGCC ACATCTGAGT CGGCCTGAGA 
               
               
                   
               
               
                 13441 
                 TAGACTTTTC CCTCTAAACT GGGAGAATAT CACAGTGGTT TTTGTTAGCA GAAAATGCAC 
               
               
                   
               
               
                 13501 
                 TCCAGCCTCT GTACTCATCT AAGCTGCTTA TTTTTGATAT TTGTGTCAGT CTGTAAATGG 
               
               
                   
               
               
                 13561 
                 ATACTTCACT TTAATAACTG TTGCTTAGTA ATTGGCTTTG TAGAGAAGCT GGAAAAAAAT 
               
               
                   
               
               
                 13621 
                 GGTTTTGTCT TCAACTCCTT TGCATGCCAG GCGGTGATGT GGATCTCGGC TTCTGTGAGC 
               
               
                   
               
               
                 13681 
                 CTGTGCTGTG GGCAGGGCTG AGCTGGAGCC GCCCCTCTCA GCCCGCCTGC CACGGCCTTT 
               
               
                   
               
               
                 13741 
                 CCTTAAAGGC CATCCTTAAA ACCAGACCCT CATGGCTGCC AGCACCTGAA AGCTTCCTCG 
               
               
                   
               
               
                 13801 
                 ACATCTGTTA ATAAAGCCGT AGGCCCTTGT CTAAGCGCAA CCGCCTAGAC TTTCTTTCAG 
               
               
                   
               
               
                 13861 
                 ATACATGTCC ACATGTCCAT TTTTCAGGTT CTCTAAGTTG GAGTGGAGTC TGGGAAGGGT 
               
               
                   
               
               
                 13921 
                 TGTGAATGAG GCTTCTGGGC TATGGGTGAG GTTCCAATGG CAGGTTAGAG CCCCTCGGGC 
               
               
                   
               
               
                 13981 
                 CAACTGCCAT CCTGGAAAGT AGAGACAGCA GTGCCCGCTG CCCAGAAGAG ACCAGCAAGC 
               
               
                   
               
               
                 14041 
                 CAAACTGGAG CCCCCATTGC AGGCTGTCGC CATGTGGAAA GAGTAACTCA CAATTGCCAA 
               
               
                   
               
               
                 14101 
                 TAAAGTCTCA TGTGGTTTTA TCTACTTTTT TTTTCTTTTT CTTTTTTTTT GAGACAAGGC 
               
               
                   
               
               
                 14161 
                 CTTGCCCTCC CAGGCTGGAG TGCAGTGGAA TGACCACAGC TCACCGCAAC CTCAAATTCT 
               
               
                   
               
               
                 14221 
                 TGCGTTCAAG TGAACCTCCC ACTTTAGCCT CCCAAGTAGC TGGGACTACA GGCGCACGCC 
               
               
                   
               
               
                 14281 
                 ATCACACCCG GCTAATTGAA AAATTTTTTT TTTTGTTTAG ATGGAATCTC ACTTTGTTGC 
               
               
                   
               
               
                 14341 
                 CCAGGCTGGT CTCAAACTCC TGGGCTCAAG TGATCATCCT GCTTCAGCGT CCGACTTGTT 
               
               
                   
               
               
                 14401 
                 GGTATTATAG GCGTGAGCCA CTGGGCCTGA CCTAGCTACC ATTTTTTAAT GCAGAAATGA 
               
               
                   
               
               
                 14461 
                 AGACTTGTAG AAATGAAATA ACTTGTCCAG GATAGTCGAA TAAGTAACTT TTAGAGCTGG 
               
               
                   
               
               
                 14521 
                 GATTTGAACC CAGGCAATCT GGCTCCAGAG CTGGGCCCTC ACTGCTGAAG GACACTGTCA 
               
               
                   
               
               
                 14581 
                 GCTTGGGAGG GTGGCTATGG TCGGCTGTCT GATTCTAGGG AGTGAGGGCT GTCTTTAAAG 
               
               
                   
               
               
                 14641 
                 CACCCCATTC CATTTTCAGA CAGCTTTGTC AGAAAGGCTG TCATATGGAG CTGACACCTG 
               
               
                   
               
               
                 14701 
                 CCTCCCCAAG GCTTCCATAG ATCCTCTCTG TACATTGTAA CCTTTTATTT TGAAATGAAA 
               
               
                   
               
               
                 14761 
                 ATTCACAGGA AGTTGTAAGG CTAGTACAGG GGATCC. 
               
             
          
         
       
     
         [0036]    The “ATG” start codon is underlined and bolded in the sequence above. See also GenBank Accession No. GenBank: U75285.1, Version: U75285.1 GI:2315862,  Homo sapiens apoptosis inhibitor survivin gene, complete cds,  and Ambrosini, et al.,  Nature Medicine,  3:917 -921 (1997), each of which is specifically incorporated by reference herein in its entirety. 
         [0037]    As illustrated in  FIG. 9 , for reference purposes, in addition to the nucleotide numbering provided herein and in the sequence listing, nucleotides beginning with, and downstream of, the “A” in a start codon can be referred to by counting positively from +1, while sequences upstream of “A” in the start codon can be referred to by counting negatively in the reverse direction from −1 beginning with the nucleotide immediately adjacent to and upstream of the start codon “A.” Thus, in the sequence above, the underlined “ATG” can be referred to as nucleotides +1, +2, +3, respectively, while the “C” adjacent to and upstream of the “ATG,” can be referred to as nucleotide −1. 
         [0038]    The disclosed constructs for use in detection and treatment of uterine cancer typically include a survivin promoter. For example, the survivin promoter can include the expression control sequence of the nucleic acid sequence of SEQ ID NO:4, or SEQ ID NO:1, illustrated in  FIG. 9 , adapted from Li and Altieri, et al.,  Biochem. J,  344:305-311 (1999), which is specifically incorporated by reference in its entirety. 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                 (SEQ ID NO: 1) 
                   
               
             
          
           
               
                    1 
                 AAATTGACAT CGGGCCGGGC GCAGTGGCTC ACATCTGTAA TCCCAGCACT TTGGGAGGCC 
                   
               
               
                   
               
               
                   61 
                 GAGGCAGGCA GATCACTTGA GGTCAGGAGT TTGAGACCAG CCTGGCAAAC ATGGTGAAAC 
               
               
                   
               
               
                  121 
                 CCCATCTCTA CTAAAAATAC AAAAATTAGC CTGGTGTGGT GGTGCATGCC TTTAATCTCA 
               
               
                   
               
               
                  181 
                 GCTACTCGGG AGGCTGAGGC AGGAGAATCG CTTGAACCCG TGGCGGGGAG GAGGTTGCAG 
               
               
                   
               
               
                  241 
                 TGAGCTGAGA TCATGCCACT GCACTCCAGC CTGGGCGATA GAGCGAGACT CAGTTTCAAA 
               
               
                   
               
               
                  301 
                 TAAATAAATA AACATCAAAA TAAAAAGTTA CTGTATTAAA GAATGGGGGC GGGGTGGGAG 
               
               
                   
               
               
                  361 
                 GGGTGGGGAG AGGTTGCAAA AATAAATAAA TAAATAAATA AACCCCAAAA TGAAAAAGAC 
               
               
                   
               
               
                  421 
                 AGTGGAGGCA CCAGGCCTGC GTGGGGCTGG AGGGCTAATA AGGCaAGGCC TCTTATCTCT 
               
               
                   
               
               
                  481 
                 GGCCATAGAA CCAGAGAAGT GAGTGGATGT GATGCCCAGC TCCAGAAGTG ACTCCAGAAC 
               
               
                   
               
               
                  541 
                 ACCCTGTTCC AAAGCAGAGG ACACACTGAT TTTTTTTTTA ATAGGCTGCA GGAUTTACTG 
               
               
                   
               
               
                  601 
                 TTGGTGGGAC GCCCTGCTTT GCGAAGGGAA AGGAGGAGTT TGCCCTGAGC ACAGGCCCCC 
               
               
                   
               
               
                  661 
                 ACCCTCCACT GGGCTTTCCC CAGCTCCCTT GTCTTCTTAT CACGGTAGTG GCCCAGTCCC 
               
               
                   
               
               
                  721 
                 TGGCCCCTGA CTCCAGAAGG TGGCCCTCCT GGAAACCCAG GTCGTGCAGT CAACGATGTA 
               
               
                   
               
               
                  781 
                 CTCGCCGGGA CAGCGATGTC TGCTGCACTC CATCCCTCCC CTGTTCATTT GTCCTTCATG 
               
               
                   
               
               
                  841 
                 CCCGTCTGGA GTAGATGCTT TTTGCAGAGG TGGCACCCTG TAAAGCTCTC CTGTCTGACT 
               
               
                   
               
               
                  901 
                 TTTTTTTTTT TTTTAGACTG AGTTTTGCTC TTGTTGCCTA GGCTGGAGTG CAATGGCACA 
               
               
                   
               
               
                  961 
                 ATCTCAGCTC ACTGCACCCT CTGCCTCCCG GGTTCAAGCG ATTCTCCTGC CTCAGCCTCC 
               
               
                   
               
               
                 1021 
                 CGAGTAGTTG GGATTACAGG CATGCACCAC CACGCCCAGC TAATTTTTGT ATTTTTAGTA 
               
               
                   
               
               
                 1061 
                 GAGACAAGGT TTCACCGTGA TGGCCAGGCT GGTCTTGAAC TCCAGGACTC AAGTGATGCT 
               
               
                   
               
               
                 1141 
                 CCTGCCTAGG CCTCTCAAAG TGTTGGGATT ACAGGCGTGA GCCACTGCAC CCGGCCTGCA 
               
               
                   
               
               
                 1201 
                 CGCGTTCTTT GAAAGCAGTC GAGGGGGCGC TAGGTGTGGG CAGGGACGAG CTGGCGCGGC 
               
               
                   
               
               
                 1261 
                 GTCGCTGGGT GCACCGCGAC CACGGGCAGA GCCACGCGGC GGGAGGACTA CAACTCCCGG 
               
               
                   
               
               
                 1321 
                 CACACCCCGC GCCGCCCCGC CTCTACTCCC AGAAGGCCGC GGGGGGTGGA CCGCCTAAGA 
               
               
                   
               
               
                 1361 
                 GGGCGTGCGC TCCCGACATG CCCCGCGGCG CGCCATTAAC CGCCAGATTT GAATCGCGGG 
               
               
                   
               
               
                 1441 
                 ACCCGTTGGC AGAGGTGGCG GCGGCGGC   AT G   GGTGCCCCG ACGTTGCCCC CTGCCTGGCA 
               
               
                   
               
               
                 1501 
                 GCCCTTTCTC AAGGACCACC GCATCTCTAC ATTCAAGAAC TGGCCCTTCT TGGAGGGCTG 
               
               
                   
               
               
                 1561 
                 CGCCTGCACC CCGGAGCGGG TGAGACTGCC CGGCC. 
               
             
          
         
       
     
         [0039]    The “ATG” start codon is underlined and bolded in the sequence above. 
         [0040]    In some embodiments, the survivin promoter includes a 260 by DNA fragment of the human survivin promoter according to nucleotides −230 to +30 of  FIG. 9  (SEQ ID NO:1), or an expression controlling fragment thereof. Nucleotides −230 to +30 of  FIG. 9  (SEQ ID NO:1) are: 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                 (SEQ ID NO: 5) 
                   
               
             
          
           
               
                   1 
                 GGCAGGGACG AGCTGGCGCG GCGTCGCTGG GTGCACCGCG ACCACGGGCA GAGCCACGCG 
                   
               
               
                   
               
               
                  61 
                 GCGGGAGGAC TACAACTCCC GGCACACCCC GCGCCGCCCC GCCTCTACTC CCAGAAGGCC 
               
               
                   
               
               
                 121 
                 GCGGGGGGTG GACCGCCTAA GAGGGCGTGC GCTCCCGACA TGCCCCGCGG CGCGCCATTA 
               
               
                   
               
               
                 181 
                 ACCGCCAGAT TTGAATCGCG GGACCCGTTG GCAGAGGTGG CGGCGGCGGC    ATG   GGTGCCC 
               
               
                   
               
               
                 241 
                 CGACGTTGCC CCCTGCCTGG. 
               
             
          
         
       
     
         [0041]    The “ATG” start codon is underlined and bolded in the sequence above. In some embodiments, the expression controlling fragment includes nucleotides −230 to −1 of  FIG. 9  (SEQ ID NO:1), which are: 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                 (SEQ ID NO: 6) 
                   
               
             
          
           
               
                   1 
                 GGCAGGGACG AGCTGGCGCG GCGTCGCTGG GTGCACCGCG ACCACGGGCA GAGCCACGCG 
                   
               
               
                   
               
               
                  61 
                 GCGGGAGGAC TACAACTCCC GGCACACCCC GCGCCGCCCC GCCTCTACTC CCAGAAGGCC 
               
               
                   
               
               
                 121 
                 GCGGGGGGTG GACCGCCTAA GAGGGCGTGC GCTCCCGACA TGCCCCGCGG CGCGCCATTA 
               
               
                   
               
               
                 181 
                 ACCGCCAGAT TTGAATCGCG GGACCCGTTG GCAGAGGTGG CGGCGGCGGC 
               
             
          
         
       
     
         [0042]    In some embodiments, the survivin promoter is derived from a homologue or orthologue of the promoter of SEQ ID NO:1 or 4. The survivin promoter can be an expression controlling nucleic acid sequence having at least 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99 percent sequence identity to the promoter of SEQ ID NO:1, or to a functional fragment thereof, such as nucleotides −230 to +30 of  FIG. 9  (SEQ ID NO:1), which is SEQ ID NO:5, or −230 to −1 of  FIG. 9  (SEQ ID NO:1), which is SEQ ID NO:6. 
         [0043]    2. Preferred Reporters Genes 
         [0044]    The disclosed constructs for detection of uterine cancer typically include a survivin promoter operably linked to a report. Suitable reporters are well known in the art, and include, but are not limited to, bacterial GUS gene, the firefly luciferase gene, and the cyan, green, red, and yellow fluorescent protein genes. These examples, are non-limiting, as the reporter can be any gene for which an easy and reliable assay is available can serve as the reporter gene. One of skill in the art knows which reporters are suitable or preferred for in vivo applications, ex vivo applications, or both. 
         [0045]    In a particular embodiment, the reporter gene is luciferase gene of pGL3/Basic (Promega, catalog number E1751), GenBank accession no. U47295.2 (Cloning vector pGL3-Basic, complete sequence), which is specifically incorporated by reference in its entirety and provides the luciferase polypeptide sequence: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 2) 
               
               
                 MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVD 
               
               
                   
               
               
                 ITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGV 
               
               
                   
               
               
                 AVAPANDIYNERELLNSMGISQPTVVEVSKKGLQKILNVQKKLPIIQKII 
               
               
                   
               
               
                 IMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSG 
               
               
                   
               
               
                 STGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGEGME 
               
               
                   
               
               
                 TTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTL 
               
               
                   
               
               
                 IDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAIL 
               
               
                   
               
               
                 ITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG 
               
               
                   
               
               
                 YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA 
               
               
                   
               
               
                 PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVD 
               
               
                   
               
               
                 YVASQVITAKKLRGGVVEVDEVPKGLTGKLDARKIREILIKAKKGGKIAV. 
               
             
          
         
       
     
         [0046]    Accordingly, in some embodiments the reporter gene includes a nucleic acid sequence encoding SEQ ID NO:2, for example, the nucleic acid sequence including nucleotides 88-1740 of GenBank accession no. U47295.2. 
         [0047]    3. Vectors 
         [0048]    The disclosed constructs including a survivin promoter can be inserted, using known methods, into any suitable expression vector. In particular preferred embodiments, the vector is recombinant adenoviral vector. Suitable adenoviral vectors and methods of cloning expression constructs into them are known in the art. 
         [0049]    In a particular embodiment, the vector is a human Ad5 adenovirus. Sequences for Ad5 are known in the art, and can be used as the backbone for survivin driven reporter constructs disclosed herein. See, for example, Genbank accession number M73260.1 (Mastadenovirus h5 gene, complete genome), which is specifically incorporated by reference herein in its entirety. In some embodiments, E1, E3, or both are deleted or substituted (He, et al.,  Proc Natl Acad Sci USA.,  95(5):2509-14 (1998)). For example, in some embodiments, an E1 deletion is a deletion of nucleotides 455-3512, or a fragment thereof. In some embodiments, an E3 deletion is a deletion of 28587-30464, or a fragment thereof. 
         [0050]    4. Method of Making Constructs and Exemplary Constructs 
         [0051]    Recombinant vectors that express a reporter operably linked to a survivin expression control sequence can be constructed according to methods that are known in the art. For example, in some embodiments, the vector includes a nucleic acid sequence encoding a survivin promoter operably linked to a nucleic acid sequence encoding a firefly luciferase open reading frame. The vector can be an adenoviral vector prepared according to known materials and methods. See, for example, the AdEasy system (Agilent Technologies, Quantum, etc.) and He, et al.,  Proc Natl Acad Sci U.S.A.,  95(5):2509-14 (1998). In a specific embodiment, a construct including a luciferase reporter gene from pGL3Basic and a simian virus 40 polyadenylation (SV40 poly-A) signal driven by a survivin promoter, are cloned into the E1-deleted region of the adenoviral vector backbone using the AdEasy system. The survivin promoter is a 260 by DNA fragment of the survivin promoter (nucleotides −230 to +30 according to Li and Altieri, et al.,  Biochem. J,  344:305-311 (1999) 
         [0052]    In particular embodiments, the survivin promoter is PCR amplified with or without 5′ and 3′ ends containing restriction sites suitable for cloning, or the fragment is excised by restriction digestion from a plasmid containing the promoter. For example, BamHI/Hind III can be used to recover the 260 by sequence from pLuc-cycl.2 (Li and Altieri, et al.,  Biochem. J,  344:305-311 (1999), which is specifically incorporated by reference herein in its entirety). The promotor can optionally be subcloned into an intermediate vector, such as PBS IISK(+) vector (Stratagene, La Jolla, Calif.), for example to utilize alternative restriction sites, or to add or subtract other expression construct elements if-needed. For example, following subcloning of BamHI/Hind III fragment of pLuc-cycl.2, a SacI/HindIII fragment can be cloned into pGL3/Basic (Promega, catalog number E1751) to a construct with a survivin promoter operably linked to a luciferase open reading frame. 
         [0053]    It will be appreciated that the foregoing is an exemplary preparation, and steps can be omitted, substituted, or added as is known in the art. For example, any of the subcloning steps can include PCR application of the desired fragment; alternative vectors can be utilized for an alternative luciferase sequences; an alternative reporter gene can be substituted for luciferase; the survivin promoter can include more or fewer nucleotides or can be an alternative promoter sequence compared to the 260 bp promoter sequence of pLuc-cycl.2 (e.g., having one or more polymorphisms, etc.); and/or other eukaryotic expression regulatory elements can be added or subtracted. 
         [0054]    Once the expression construct is a prepared, it can be inserted into an adenoviral expression system. In some embodiments, the construct is subcloned into shuttle vector (e.g., pShuttle vector (Quantum, Montreal, Quebec, Canada)), and cloned into adenoviral expression system by homologous recombination, though alternative methods of preparing adenoviral vectors well known and can be substituted. In the specific embodiment described above, a KpnI/SalI fragment from pGL3B Survivin can be subcloned into pShuttle vector to create pShuttleGL3BSurvivin and homologous recombination can be performed in BJ5183 cells in accordance with the AdEasy System to create a recombinant adenovirus, ( FIG. 10 ), in which the luciferase reporter expression is driven by the survivin promoter. See also, Zhu, et al.,  Cancer Gene Therapy,  11:256-262 (2004), and Houdt, et al.,  J. Neurosurg.,  104:583-592 (2006). 
         [0055]    Other materials for preparing the disclosed constructs are also available and include, for example, BIRC5 (NM_001168) Human cDNA ORF Clone (Origene Catalogue Number RC205935), which is a BIRC5 (Myc-DDK-tagged)-Human baculoviral IAP repeat containing 5 (BIRC5/Survivin), transcript variant 1; pBS Survivin (Addgene Plasmid #19233), which is a pBluescript vector backbone including a  G. gallus  (chicken) survivin insert according to GenBank: FG356243.1 (PC/PO 2-94 Embryonic chicken perichondrium/periosteum library  Gallus gallus  cDNA clone PC/PO 2-94 5-similar to Baculoviral IAP repeat-containing 5 (survivin) (BIRC5), transcript variant 1, mRNA sequence) and Gene ID: 374078 (BIRC5 baculoviral IAP repeat containing 5 [ Gallus gallus  (chicken)]), each of which are specifically incorporated by reference in their entireties; and Luciferase-pcDNA3 (Addgene Plasmid #18964) which is a pcDNA3 including a Firefly Luciferase insert. Nucleic acid and protein sequences of all of the foregoing references, accession numbers, and reagents (e.g., plasmids and other vectors) are specifically incorporated by reference in their entireties. 
         [0056]    B. Constructs for Treating Uterine Cancer 
         [0057]    Constructs for treating uterine cancer include expression vectors under the control of a survivin promoter. The vectors can be prepared using materials and methods including those described above for the report constructs and others known in the art. However, it will be appreciated the therapeutic constructs optionally include a reporter gene (e.g., a reporter is not required for therapeutic constructs), and may further include additional elements not present in reporter constructs. The expression vectors can encode tumor suppressor genes, cytotoxic genes, cytostatic genes, cytokines, suicide genes, oncolytic virus and antigen-encoding genes. Examples of tumor suppressor genes include WT1, p53, p16, Rb, BRCA1, and LK8. 
         [0058]    One embodiment provides a construct that expresses an oncolytic virus under the control of a survivin promoter. Oncolytic virus (OV) therapy is based on selective replication of viruses in cancer cells and their subsequent spread within a tumor without causing damage to normal tissue. Typically, OVs fall into two classes: (i) viruses that naturally replicate preferentially in cancer cells and are nonpathogenic in humans often due to elevated sensitivity to innate antiviral signaling or dependence on oncogenic signaling pathways. These include autonomous parvoviruses, myxoma virus (MYXV; poxvirus), Newcastle disease virus (NDV; paramyxovirus), reovirus, and Seneca valley virus (SVV; picornavirus); and (ii) viruses that are genetically manipulated for use as vaccine vectors, including measles virus (MV; paramyxovirus), poliovirus (PV; picornavirus), and vaccinia virus (VV; poxvirus), and/or those genetically engineered with mutations/deletions in genes required for replication in normal but not in cancer cells including adenovirus (Ad), herpes simplex virus (HSV), VV, and vesicular stomatitis virus (VSV; rhabdovirus). 
         [0059]    C. Pharmaceutical Compositions 
         [0060]    The nucleic acid constructs can be combined with a pharmaceutically acceptable excipient or carrier to form a pharmaceutically acceptable composition. Examples of suitable excipients or carriers include but are not limited to water, salt water, alcohol, lipid, wax, buffer solution, solid carrier such as mannitol, lactose, starches, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate, or biodegradable microsphere (e.g., polylactate polyglycolate). 
         [0061]    The compositions may be provided in the form of single dose or multi-dose container such as sealed ampule or vial. Preferably, such container may be sealed so as to conserve aseptic condition of pharmaceutical formulations before using. In general, the formulation may be preserved as suspension, fluid, and emulsion in oil or aqueous vehicle. Further, the pharmaceutical formulation may be preserved under freeze drying conditions. 
         [0062]    The pharmaceutical compositions may be administered with site-specific injection or intravenous injection. Site-specific injection includes, for example, intraperitoneal injection, intrapleural injection, intrathecal injection, intraarterial injection, intratumoral injection or local application. The preferred method is intravenous injection. 
         [0063]    It should be understood that the suitable amount of the nucleic acid construct actually administered ought to be determined in light of various relevant factors including the condition to be treated, the age and weight of the individual patient, food, administration time, excretion rate, the severity of the patient&#39;s symptom and reaction susceptibility; and, therefore, the above dose should not be intended to limit the scope of the invention in any way. Generally, the adenoviral vector contained in the pharmaceutical composition may be administered in an appropriate physiologically acceptable carrier at a dose of about 10 4  to about 10 14  vp/mL. The multiplicity of infection may be generally in the range of 0.001 to 100, preferably 5, 10, 20, or 50. If administered as a polynucleotide construct, about 0.01 to 1000 μg/kg of an adenoviral vector can be administered. The adenoviral vector may be administered one or more time, depending upon the intended use and the immune response potential of the host, and may also be administered as multiple, simultaneous injections. If an immune response is undesirable, the immune response may be diminished by employing a variety of immunosuppressants, or by employing a technique such as an immunoadsorption procedure (e.g., immunoapheresis) that removes adenovirus antibody from the blood, so as to permit repetitive administration, without a strong immune response. 
         [0064]    The composition may be used as the single therapy. But it may be combined with other anti-tumor protocols, such as conventional chemotherapy or radiation therapy for treating cancer. The chemotherapy drug which can be used with composition of the present invention encompasses paclitaxel, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate. The radiation therapy which can be used with the composition of the present invention may be X-ray irradiation and .gamma.-ray irradiation, etc. 
         [0065]    The adenovirus produced from the adenoviral vector shows high oncolytic effect in tumor cells, while low effect in normal cells from in vitro and in vivo experiments. Thus, the viral vector comprising the tumor-specific promoter may be used for treating a cancer. 
       III. Methods for Detecting Uterine Cancer 
       [0066]    One method for detecting uterine cancer includes contacting a cell or cells suspected of being cancerous with a vector containing a reporter gene, wherein the expression of the reporter gene is under the control of a survivin promoter. In a preferred embodiment, the vector is an adenoviral vector and the reporter gene encodes luciferase. 
         [0067]    Another method provides administering to a subject suspected of having uterine cancer, an effective amount of a vector containing a reporter gene, wherein expression of the reporter gene is under the control of a survivin promoter. The vector can be administered systemically or directly into the uterus. 
         [0068]    One embodiment provides a method for distinguishing cancerous uterine cells from non-cancerous uterine cells by contacting a population of uterine cells containing a mixture of cancerous and non-cancerous uterine cells with a vector containing a reporter gene, wherein the expression of the reporter gene is under the control of a survivin promoter, and detecting expression of the reporter gene wherein, expression of the reporter gene indicates that the cell expressing the reporter gene is cancerous. The contacting can be in vivo or ex vivo. Accordingly, methods of detecting uterine cancer in the subject and in tissue samples isolated from a subject are both specifically disclosed. 
       IV. Methods for Treating Uterine Cancer 
       [0069]    One method for treating uterine cancer includes contacting a cell or cells suspected of being cancerous with a vector containing a gene that encodes tumor suppressor genes, cytotoxic genes, cytostatic genes, cytokines, suicide genes, oncolytic virus and antigen-encoding genes. Examples of tumor suppressor genes include WT1, p53, p16, Rb, BRCA1, and LK8. The expression of the gene is under the control of a survivin promoter. In a preferred embodiment, the vector is an adenoviral vector encoding an oncolytic virus, wherein the expression of the oncolytic virus is under the control of a survivin promoter. 
         [0070]    Another method provides administering to a subject suspected of having uterine cancer, an effective amount of a vector encoding a cytotoxic agent, wherein expression of the cytotoxic agent is under the control of a survivin promoter. The vector can be administered systemically or directly into the uterus. 
       EXAMPLES 
     Example 1 
     X-Gal Staining 
       [0071]    Materials and Methods 
         [0072]    All procedures performed on animals were approved by Georgia Regents University&#39;s Institutional Animal Care and Use Committee and were within the guidelines of humane care of laboratory animals. Materials and methods used in viral vector construction, cell culture, and in vitro transfection experiments, s.c. Tumor and intrauterine models, and local vector administration, plasma collection, and statistical analyses are detailed herein materials and methods. 
       Recombinant Adenovirus and Promoters 
       [0073]    Large-scale production of adenovirus vectors was performed as we have described previously with a typical batch yield of 2×10 10  plaque-forming units (PFU)/ml (Al-Hendy, A., et al., Am J Obstet Gynecol, 182(3): 553-559 (2000)). Ad vectors used in this study are listed in Table 1. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Description of the Adenovirus vectors used in this study 
               
             
          
           
               
                   
                   
                   
                 Modification 
                   
               
               
                 No. 
                 virus 
                 Promoter 
                 Site 
                 Virus description 
               
               
                   
               
               
                 1 
                 Ad5-luc 
                 CMV 
                 WILD 
                 E1/E3 deleted, a 
               
               
                   
                   
                   
                   
                 luciferase gene under the 
               
               
                   
                   
                   
                   
                 CMV promoter in place of 
               
               
                   
                   
                   
                   
                 E1 
               
               
                 2 
                 Ad5-LacZ 
                 CMV 
                 WILD 
                 E1/E3 deleted, a luciferase 
               
               
                   
                   
                   
                   
                 gene under the CMV 
               
               
                   
                   
                   
                   
                 promoter in place of E1 
               
               
                 3 
                 Ad5-survivin-luc 
                 Survivin 
                 Promoter 
                 E1/E3 deleted, a luciferase 
               
               
                   
                   
                   
                   
                 gene under the Survivin 
               
               
                   
                   
                   
                   
                 promoter in place of E1 
               
               
                 4 
                 Ad5-heparanase- 
                 Heparanase 
                 Promoter 
                 E1/E3 deleted, a luciferase 
               
               
                   
                 luc 
                   
                   
                 gene under the heparanase 
               
               
                   
                   
                   
                   
                 promoter in place of E1 
               
               
                 5 
                 Ad5-SLPI-uc 
                 Secretory 
                 Promoter 
                 E1/E3 deleted, a luciferase 
               
               
                   
                   
                 leukoprotease 
                   
                 gene under the SLPI 
               
               
                   
                   
                 Inhibitor 
                   
                 promoter in place of E1 
               
               
                   
                   
                 (SLPI) 
               
               
                   
               
               
                 No. 1 is described in Krasnykh, V. N., et al., J Virol 70(10): 6839-6846 (1996). 
               
               
                 No. 2 is described in Franklin, R., M. Quick and G. Haase, Gene Ther 6(8): 1360-1367 (1999). 
               
               
                 No. 3 is described in Van Houdt, W. J., et al., J Neurosurg 104(4): 583-592 (2006). 
               
               
                 No. 4 is described in Breidenbach, M., et al., Cancer Lett 240(1): 114-122 (2006). 
               
               
                 No. 5 is described in Barker, S. D., et al., Gene Ther 10(14): 1198-1204 (2003).  Cell cultures   
               
             
          
         
       
     
         [0074]    For experimental models, the human leiomyosarcoma cell line SKUT-1 was used and purchased from American type Culture collection (ATCC® HTB-114™) that is derived from 75 years old Caucasian female originating from grade III, mesodermal tumor (mixed); consistent with uterine leiomyosarcoma. 
         [0075]    The cells are considered biosafety level 1 based on U.S. Public Health Service Guidelines. Primary cultures of human leiomyoma cells were derived from fibroid tumors removed during hysterectomies. Human leiomyoma tissues were collected according to the policies of the Institutional Review Board of Georgia Regents University, Augusta, Ga., USA, and used to establish primary fibroid (1ry F) cells, as described previously (Rauk, P. N., et al, Am J Obstet Gynecol 173(2): 571-577 (1995); Al-Hendy, A., et al., Am J Obstet Gynecol 191(5): 1621-1631 (2004)). To represent normal cells (controls), we used a human myometrial cell line (Myo N); this cell line was cultured and maintained as described previously (Carney, M. E. Hawaii Med J 61(12): 283-286 (2002)). 
       X Gal Staining of Fixed Leiomyosarcoma Cells 
       [0076]    This experiment was done to evaluate the susceptibility of SKUT-1 cells to transfection by wild type Adenovirus serotype 5 with B-Galactosidase as a reporter gene. Three different multiplicities of infection (MOI) of 1, 3, and 5 were used. The viral particles were mixed with cell culture media followed by 1 hour of mild shaking then regular cell culture conditions were applied. 24 hours later, X-Gal staining was performed on the cells. 
         [0077]    Results 
         [0078]    Human Leiomyosarcoma cells (LMS) are susceptible to wild type adenovirus transfection by X-Gal Staining. The transgene of the bacterial enzyme β-galactosidase can be easily located with a LacZ stain using the artificial substrate X-Gal, which turns blue when it is cleaved by B-Galactosidase. Culture media was aspirated off and washed SKUT-1 cells 1× with cold PBS, fixed the cells on ice with ˜5 mL glutaraldehyde (1:100 dilution of stock in PBS) for 5 min, rinse the cells 3× with PBS for 4 min. per wash, dilute 25× stock of X-Gal into the staining solution (final 1 mg/mL of X-gal), add ˜5 mL of the X-gal staining solution to the cells and incubate at 37° C. for 24 hours. Cells were checked every 4 hours to determine whether cells were turning blue. ( FIGS. 1A-1C ) Transfection of Leiomyosarcoma cells by Ad-lacZ, X-Gal staining of human SK-UT 1 cells after transfection with Adenovirus with Ad-Lac Z reporter gene at multiplicity of infection 1 MOI ( FIG. 1A ), 3 MOI ( FIG. 1B ) and 5 MOI ( FIG. 1C ). X-gal is an analogue of lactose and therefore hydrolyzed by the β-galactosidase enzyme giving intensely blue products. 
       Example 2 
     Screening for Sarcoma Specific Gene Expression 
       [0079]    Materials and Methods 
       Luciferase Assay 
       [0080]    To screen the 3 promoters of interest for their sarcoma specific gene expression potential, #3 60 mm 2  cell culture dishes of SKUT-1 cells at 70% confluence were transfected with 3 different adenoviral constructs which are Ad Survivin, Ad Heparanase, and Ad SLPI all at the same MOI the same technique as described above and used luciferase assay to differentiate between gene expression levels under the control of the 3 used promoters. Growth medium was removed from cultured cells, and the cells were rinsed in 1× PBS. Then without dislodging cells, as much of the final wash was removed as possible. 400 ul volume of 1× cell lysis buffer was dispensed (CCLR) into each culture vessel then attached cells were scraped from the dish, and the cells and solution were transferred to a microcentrifuge tube. Debris was separated by brief centrifugation, and the supernatant was transferred to a new tube. 20 μl of cell lysate was used with 100 μl of luciferase assay reagent and measured the light produced by Synergy HT microplate reader utilizing Gen-5 software for bioluminescence detection. The classic luciferase assay was used to compare the degree of reporter gene expression in leiomyosarcoma cells under different promoters. 
         [0081]    Results 
         [0082]    Screening of reporter gene expression in LMS cells under different promoters reveals Ad-Survivin as a malignancy specific promoter “Luciferase assay.”  FIG. 2  shows the results of the luciferase assay in Leiomyosarcoma tissue, primary fibroid tissue, and myometrium transfected with constructs having a Survivin promoter at 1 MOI, Survivin promoter at 5 MOI, Secretory Leukoprotease Inhibitor promoter at 1 MOI, Secretory Leukoprotease Inhibitor promoter at 5 MOI, Heparanase promoter at 1 MOI, Heparanase promoter at 5 MOI. 
       Example 3 
     In Vitro Bioluminescence Imaging 
       [0083]    Materials and Methods 
       Animal Model 
       [0084]    SKUT-1, 1ry F, Myo N cells (5×10 6  or 20×10 6 ) were either implanted directly or transfected with Adenovirus first then implanted into the right flank of female nude mice (Nu/Nu; Harlan Laboratory), and tumors developed over a period of 3-5 days. 
       Luciferin D In Vitro Bioluminescence Imaging 
       [0085]    To confirm the superior Ad Survivin controlled reporter gene expression by IVIS live cell-imaging, the same number of cells were transfected and imaged using Xenogen IVIS 100 (Caliper Life Sciences, Hopkinton, Mass.), an optical imaging device with extremely light-tight, low background imaging chamber. The device was used for in vitro, ex vivo, and in vivo bioluminescence detection. Firefly D-Luciferin 15 mg/ml in PBS was added to cell culture media in culture dishes. The software mode was set to luminescence, photography and X-ray. Exposure was 600 with low binning (Henriques, Henriques-Pons, et al. 2014). SKUT-1 Cells are highly expressing luciferase enzyme under survivin promoter in comparison to benign tumor cells as well as healthy myometrial cells in vitro by “IVIS Live cell Bioimaging” 
         [0086]    Results 
         [0087]    SKUT-1 Cells highly express luciferase enzyme under survivin promoter control in comparison to benign tumor cells as well as healthy myometrial cells in vitro by “IVIS Live cell Bioimaging” ( FIG. 3 ). The order of dishes is Leiomyosarcoma, Primary Fibroid and Myometrium respectively.  FIG. 4  shows the correlation of the total photon emission/second to the MOI of the different studied cell lines. 
       Example 4 
     Ex Vivo and In Vivo Bioluminescence Imaging 
       [0088]    Materials and Methods 
       Luciferin-D Ex Vivo and In Vivo Bioluminescence Imaging 
       [0089]    Either ex vivo or in vivo transfection was utilized in animal studies. In the ex vivo case, transfected cells were implanted in the animals either subcutaneously or intrauterine. Untransfected cells were implanted in the nude mice and then, when the tumor developed the virus was injected intravenously. D-Luciferin 15 mg/ml in PBS was intraperitoneally injected at 260 ul per mouse followed by isofluran inhalation anesthesia 10 minutes later. The anesthetized animals were then placed in the IVIS chamber. The software mode was set to luminescence, photography and X-ray. Exposure was 600 with low binning. (Henriques, C., et al., Parasit Vectors 7: 89 (2014)). 
         [0090]    Results 
         [0091]    Ex vivo bioimaging of subcutaneously injected cells shows higher expression in LMS compared to benign and normal cells ( FIGS. 5A and 5B ). Ex-vivo subcutaneous model Bioluminescence imaging at 5×10 6  cells per animal thirty minutes ( FIG. 5A ) and one hour ( FIG. 5B ) post D-luciferin injection. Showing signal only in the transfected LMS lesions and is near zero in the non-transfected as well as the begin leiomyoma case. (P&lt;0.0001) 
         [0092]    In vivo Bioimaging showing Leiomyosarcoma lesions emits significantly higher luminescence compared to benign and normal tissue ( FIGS. 6A-6C ). Bioluminescence imaging subcutaneously injected with 20×10 6  cells per animal transfected with Ad5-Survivin-luc. Signal shows only in the transfected LMS lesions and is near zero in the non-transfected as well as the begin leiomyoma case. (P&lt;0.0001). 
         [0093]      FIGS. 7A-7B  show images of mice with 20×10 6  cells injected intracervically. The cells are t-LMS, t-1ryF, or LMS cells.  FIG. 7A  is shows images thirty minutes post injection.  FIG. 7B  shows images one hour post injection, and  FIG. 7C  shows images post cellular implantation with the same number of cells.  FIG. 7D  is line graph of total photon emission per second versus time for from top to bottom t-LMS, t-1ryF, and LMS cells. 
         [0094]      FIG. 8  is an image of mice injected with 5×10 6  cells intracervically. The cells are t-LMS, t-LMS, LMS, t-1ryF, and t-1ryF cells from left to right. Signal shows only in the infected LMS lesions and is near zero in the non-transfected as well as benign leiomyoma case. 
         [0095]    While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been put forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention. 
         [0096]    All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.