臺灣博碩士論文加值系統

假性狂犬病毒(pseudorabies virus, PrV)屬於豬隻的一種疱疹病
毒。此病毒具有廣泛性的自然宿主，包含許多的哺乳類動物，但對人類是一種非致病性的病毒。在本實驗室先前的研究中，利用簡單的同源重組的方式，將假性狂犬病毒改造成gD/gE/TK缺陷的病毒來做疫苗基因的傳送載體。在治療癌症的方法中，已經有研究指出利用抑制血管新生來抑制腫瘤的生長以及利用病毒會專一性在腫瘤細胞中複製來殺死腫瘤細胞的特性來治療腫瘤。因此在這建立好的系統中，第一部分利用同源重組的方式將會抑制血管新生的endostatin基因送入假性狂犬病毒載體中，並將此病毒命名為YP1，進一步探討抑制小鼠腦瘤發展的情形。實驗結果顯示YP1確實帶有endostatin的基因，並且可表現具有生物活性的蛋白質來抑制內皮細胞的增生。在小鼠腦瘤的動物模式中，也發現在經由YP1治療的小鼠其腫瘤的生長速度比對照組來得慢，且存活也比對照組來得好。在第二個部分中，有研究指出Her2/neu蛋白質在許多的腫瘤細胞中有過度表現的情形，這些腫瘤包含有肺癌、乳癌、口腔癌以及膀胱癌。因此我們利用具有選擇性複製的病毒來殺死這些Her2/neu過度表現的腫瘤細胞。經由同源重組的方式，我們重組出帶有Her2/neu啟動子的病毒，並將此病毒命名為YP2。在實驗的結果中得知YP2可以將Her2/neu過度表現的
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CF-1、HCDB-1以及TSGH8301腫瘤細胞殺死，但對於Her2/neu不表現的細胞則不會有任何細胞病變的產生。在膀胱癌的小鼠模式中，我們連續七次將YP2病毒打入腫瘤細胞中進一步觀察其抑制腫瘤細胞生長的情形。結果顯示YP2治療的小鼠其腫瘤明顯比對照組來得小，而且存活也有明顯的延長。因此由以上的實驗證實經由同源重組的YP1和YP2兩種病毒確實有抑制小鼠腫瘤生長的情形，而且也可以增加小鼠的存活時間。假性狂犬病毒對人類是一種非致病性的病毒，而且經由簡單的同源重組的方法我們可以植入任何有興趣的治療基因。因此或許可以將假性狂犬病毒當作是一個更具有安全性
的病毒載體來治療癌症。

Pseudorabies virus (PrV), a herpesvirus of swine, is a neurotropic herpesvirus with a wide host-range, including several mammalian species, but is non-pathogenic for humans. We have previously constructed a gD/gE/TK-negative PrV mutant, which can be used as a viral vector for gene transfer. Targeting tumor angiogenesis and replication-selective oncolytic virus have been reported to kill tumor cells. In this study, we constructed a PrV vector carrying endostatin gene, the most potent inhibitor of tumor angiogenesis, for the treatment of brain tumor. Besides, previous studies indicated that Her2/neu is overexpressed in many human cancers, including lung, breast, oral and bladder cancers. Using homologous recombination method, PrV-CD5-ES and PrV-Her2P oncolytic virus were generated, and designated YP1 and YP2. In in vitro study, the expression of endostatin was detected by RT-PCR and its biological activity was determined by endothelial cell proliferation assay after YP1 infection. In C6 glioma-bearing SCID mice, the tumor in YP1-treated mice was significant decreased and survival was also prolonged. The YP2 virus could lyse CF-1, HCDB-1 and TSGH8301 cells, which were Her2/neu-overexpressing cancer cells, but not NIH3T3 cells without Her2/neu expression. In in vivo study, the YP2 virus could effectively inhibit tumor growth in mice bearing bladder cancer and survival was significant enhanced in YP2-treated mice. Because PrV is a non-pathogenic virus for humans, these viruses may serve as safer viral vectors for cancer gene therapy.

Contents
Abstract…………………………………………………………………….. I
Chinese abstract………………………………………………………….…. II
Acknowledgement……………………………………………………….…. IV
Contents………………………………………………………………….…. V
Figure contents……………………………………………………………... VIII
Abbreviation……………………………………………………………….. X
Introduction Gene therapy………………………………………………………….. 1
Pseudorabies virus………………………………………………….…. 2
Angiogenesis and endostatin……………………………………….…. 3
Oncolytic virus………………………………………………………... 3
Brain tumor….………………………………………………………… 4
Her2/neu and bladder cancer……………………………………….…. 4
The aims for cancer gene therapy…………………………………….. 5
Materials and methods
A. Mice, cells and viruses……………………………………………….. 6
B. Construction of plasmid………………………………………………. 6
C. Construction of gD-negative PrV…………………………………….. 7
D. Detection of HSV-TK, endostatin, IRES and PrV-gD genes in
recombinant PrV by PCR…………………………………………….8
E. Detection of the endostatin mRNA by RT-PCR……………………… 9
F. Proliferation assay…………………………………….…………….… 9
G. Immunohistochemistry………...……………………………………... 10
H. Assays of cytopathic effect (CPE), cell viability and viral
replication……………………………………………………………... 11
I. Luciferase assay …………………………………………………….… 11
J. Cytotoxicity assay in vitro.…………………………………………… 12
K. Animal studies………………………………………………………... 13
L. Statistical analyses…………………………………………………… 13
Results I
1. Construction of pBT/DgD-CD5-ES plasmid……………………….… 14
2. Construction of gD-negative PrV virus…………………………….…. 14
3. Detection of HSV-TK and endostatin genes in recombinant PrV by
PCR……………………………………………………………………15
4. Detection of the endostatin mRNA by RT-PCR……………………… 15
5. Detection of the endostatin protein expression……………………….. 15
6. Functional assay — cell proliferation assay……………………………. 16
7. Suppression of C6 brain tumor growth by intratumoral injection of
YP1 virus………………………………………………………………17
Results II
1. Construction of pBT/Her2P-gD-IRES-TK plasmid…………………... 18
2. Construction of gD-negative PrV virus…………………………….…. 18
3. Detection of HSV-TK, IRES and PrV-gD genes in recombinant PrV by PCR…………………19
4. The cytopathic effect and cell viability induced by YP2 virus……….. 19
5. The Her2/neu promoter was active in bladder and oral cancer cells….. 20
6. In vivo antitumor effect of YP2 virus in mice bearing bladder tumor… 21
7. Induction of cytotoxic activities in mice
bearing bladder tumor treated with YP2 virus………………21
Discussion………………………………………………………………….. 22
References………………………………………………………………….. 28
Figure……………………………………………………………………….. 32
Appendix I Genome of Pseudorabies Virus………………………………… 56
Appendix II. The strategy of generation of recombinant PrV……………………………… 57

Boldogkoi, Z., Bratincsak, A. & Fodor, I. 2002. Evaluation of
pseudorabies virus as a gene transfer vector and an oncolytic agent for
human tumor cells. Anticancer Res. 22, 2153-2159.
Boldogkoi, Z. & Nogradi, A. 2003. Gene and cancer therapy─
pseudorabies virus: a novel research and therapeutic tool? Curr. Gene
Ther. 3, 155-182.
Braz, J., Beaufour, C., Coutaux, A., Epstein, A. L., Cesselin, F., Hamon, M. and Pohl, M. 2001. Therapeutic efficiency in experimental
polyarthritis of viral-driven enkephalin overproduction in sensory
neurons. J. Neurosci. 21, 7881-7888.
Chen, Q. R., Zhang, L., Gasper, W. & Mixson, A. J. 2001. Targeting
tumor angiogenesis with gene therapy. Mol. Genet. Metab 74, 120-127.
Citri, A., Skaria, K. B. & Yarden, Y. 2003. The deaf and the dumb:
the biology of ErbB-2 and ErbB-3. Exp. Cell Res. 284, 54-65.
Cozzi, P. J., Malhotra, S., McAuliffe, P., Kooby, D. A., Federoff, H. J., Huryk, B., Johnson, P., Scardino, P. T., Heston, W. D. and Fong, Y. 2001. Intravesical oncolytic viral therapy using attenuated, replication-competent herpes simplex viruses G207 and Nv1020 is effective in the treatment of bladder cancer in an orthotopic syngeneic model. FASEB J. 15, 1306-1308.
Cozzi, P. J., Burke, P. B., Bhargav, A., Heston, W. D., Huryk, B., Scardino, P. T. and Fong, Y. 2002. Oncolytic viral gene therapy for prostate cancer using two attenuated, replication-competent, genetically engineered herpes simplex viruses. Prostate. 53, 95-100.
Doronin, K. 2001. Tissue-specific, tumor-selective, replication -competent adenovirus vector for cancer gene therapy. J. Virol. 75, 3314-3324.
28
Galanis, E., Vile, R. & Russell, S. J. 2001. Delivery systems intended for in vivo gene therapy of cancer: targeting and replication competent viral vectors. Crit Rev. Oncol. Hematol. 38, 177-192.
Gupta, N. 2000. Current status of viral gene therapy for brain tumours. Expert. Opin. Investig. Drugs 9, 713-726.
Hsieh, J. L., Wu, C. L., Lee, C. H. and Shiau, A. L. 2003. Hepatitis B virus x protein sensitize hepatocellular carcinoma cells to cytolysis induced by E1B-deleted adenovirus through the disruption of p53 function. Clinical Cancer Research. 9, 338-345.
Jolly, D. 1994. Viral vector systems for gene therapy. Cancer Gene Ther. 1, 51-64.
Klonjkowski, B., Gilardi-Hebenstreit, P., Hadchouel, J., Randrianarison, V., Boutin, S., Yeh, P., Perricaudet M. and Kremer, E. J. 1997. A recombinant E1-deleted canine adenoviral vector capable of transduction and expression of a transgene in human-derived cells and in vivo. Hum. Gene Ther. 8, 2103-2115.
Kong, H. L., Hecht, D., Song, W., Kovesdi, I., Hackett, N. R., Yayon, A., and Crystal, R. G. 1998. Regional suppression of tumor growth by in vivo transfer of a cDNA encoding a secreted form of the extracellular domain of the flt-1 vascular endothelial growth factor receptor. Human Gene Ther. 9, 823-833.
Lee, S. J. et al. 2002. Novel prostate-specific promoter derived from PSA and PSMA enhancers. Mol. Ther. 6, 415-421.
Longnecker, R., Roizman, B. and Meigner, B. 1988. Herpes simplex viruses as vectors: properties of a prototype vaccine strain suitable for use as a vector, in Gluzman, Y., Hughes, S. H. Viral vectors. Cold Spring Harbor Laboratory Press. P.68.
Lin, S. C. et al. 2000. Establishment and characterization of a cell line (HCDB-1) derived from a hamster pouch carcinoma induced by DMBA and Taiwanese betel quid extract. Proc. Natl. Sci. Counc. 24,
29
129-135.
Moriuchi, S. et al. 2000. HSV vector cytotoxicity is inversely correlated with effective TK/GCV suicide gene therapy of rat gliosarcoma. Gene Ther. 7, 1483-1490.
Mellon, J. et al. 1996. C-erbB-2 in bladder cancer: molecular biology, correlation with epidermal growth factor receptors and prognostic value. The Journal of Urology. 155, 321-326.
Natsume, A. et al. 2002. Bcl-2 and GDNF delivered by HSV-mediated gene transfer after spinal root avulsion provide a synergistic effect. J. Neurotrauma. 19, 61-68.
Natsume, A. et al. 2002. Bcl-2 and GDNF delivered by HSV-mediated gene transfer act additively to protect dopaminergic neurons from 6-OHDA-induced degeneration. Exp. Neurol. 169, 231-238.
Neal, D. E. & Mellon, K. 1992. Epidermal growth factor receptor and bladder cancer: a review. Urol. Int. 48, 365-371.
O''Reilly, M. S. et al. 1997. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-285.
Peeter, B., Jan, P., Gielkens, A. and Moormann, R. 1993. Envelope glycoprortein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice. J. Virol. 67, 170-177.
Prieto, J., Solera, J. & Tabares, E. 2002. Development of new expression vector based on Pseudorabies virus amplicons: application to human insulin expression. Virus Res. 89, 123-129.
Randazzo, B. P. et al. 1995. Treatment of experimental intracranial murine melanoma with neuroattenuated herpes simplex virus 1 mutant. Virology. 211, 94-101.
Samoto, K. et al. 2002. A herpes simplex virus type 1 mutant with gamma 34.5 and LAT deletions effectively oncolyses human U87
30
glioblastomas in nude mice. Neurosurgery 50, 599-605.
Steneson, A. J., cooper, M., Griffiths, J. C., Gibson, P. C., Whitehouse, A., Jones, E. F., Markham, A. F., Kinsey, S. E. and Meredith, D. M. 1999. Assessment of Herpesvirus saimiri as a potential human gene therapy vector. J. Med. Virol. 57, 269-277.
Shiau, A. L., Liu, C. W., Wang, S. Y., Tsai, C. Y. & Wu, C. L. 2002. A simple selection system for construction of recombinant gD-negative pseudorabies virus as a vaccine vector. Vaccine 20, 1186-1195.
Tenser, R. B., Hay, K. A. and Edris, W. A. 1989. Latency-associated transcript but not reactivatable virus is present in sensory ganalion neurons after inoculation of thymidine kinase-negative mutants of herpes simplex virus type 1. J. Virol. 63: 2861-2865.
Watthen, M. W. and Wathen, L. M. K. 1986. Characterization and mapping of a nonessential pseudorabies virus glycoprotein. J. Virol. 58: 173-178.
Wein, L. M., Wu, J. T. & Kirn, D. H. 2003.Validation and Analysis of a Mathematical Model of a Replication-competent Oncolytic Virus for Cancer Treatment: Implications for Virus Design and Delivery. Cancer Res. 63, 1317-1324.