Tumor-specific gene therapy for uterine cervical cancer using MN/CA9-directed replication-competent adenovirus Ho Yeong Lim, 1 Miwon Ahn, 2 Hyun Cheol Chung, 3 Thomas A Gardner, 4 Chinghai Kao, 4 Sang-Jin Lee, 4 and Se Joong Kim 2 1 Department of Medical Oncology; 2 Department of Urology, Ajou University School of Medicine, Suwon, Korea; 3 Cancer Metastasis Research Center, Yonsei University College of Medicine, Seoul, Korea; and 4 Department of Urology, Indiana University School of Medicine, Indianapolis, Indiana, USA. Although gene therapies using tissue-specific promoters have been reported to be a promising tool for treating cancers, few studies have explored this possibility for uterine cervical cancer. MN/CA9 is a transmembrane glycoprotein that was first identified in the human cervical carcinoma cell line, HeLa. Since MN/CA9 protein is highly expressed in uterine cervical cancer tissues, but not in normal cervix, we constructed a tumor-specific replication-competent adenoviral vector utilizing MN/CA9 promoter (Ad-MN/CA9- E1a), which can replicate only in MN/CA9-expressing cells. Infection of Ad-MN/CA9-E1a to MN/CA9-positive uterine cervical cancer cells (HeLa, C-33 A and SiHa) resulted in much stronger Ad5 E1a protein expressions compared with MN/CA9-negative cells (SK-RC-29), suggesting a tissue-specific replication of this recombinant adenovirus. In vitro cytotoxicity assay revealed that the growth of MN/CA9-positive cells was significantly inhibited with 0.01–1 MOI of Ad-MN/CA9-E1a, but the growth of MN/CA9- negative cells (SK-RC-29) could only be inhibited by as many as 100 MOI. Intratumoral injection of Ad-MN/CA9-E1a effectively induced growth delay of HeLa tumors in nude mice. These results suggest that a novel replication-competent adenoviral vector mediated by MN/CA9 promoter, Ad-MN/CA9-E1a, can selectively replicate in MN/CA9-expressing tumors with cytotoxic effects and may be utilized for the treatment of uterine cervical cancer. Cancer Gene Therapy (2004) 11, 532–538. doi:10.1038/sj.cgt.7700732 Published online 28 May 2004 Keywords: cervix neoplasms; adenovirus; virus replication; MN/CA9 I n parallel with the development of highly sensitive cytologic screening methods, comprehensive screening programs have allowed early detection and high cure rates in uterine cervical cancer in western countries. However, uterine cervical cancer is still the leading cause of cancer deaths for women in many developing countries. 1,2 The choice of treatment can be decided by several factors, including stage, histological features and the presence of lymph node metastasis. In general, intrae- pithelial lesions are treated with superficial ablative techniques and early invasive cancers are removed with surgery or radiotherapy. Although local treatments result in high cure rates in these stages of disease, some patients suffer from treatment complications such as ureterova- ginal fistula, urinary tract infection, wound infection or bladder dysfunction. 3–5 These complications compromise patients’ quality of life, despite significant improvement in survival with local treatments. Furthermore, in contrast to the high response rates of localized cervical cancer to treatments, patients with disseminated disease almost always have dismal results. With rarity, metastatic cervical cancer may respond to chemotherapy, but the response duration is usually very short and its adverse effects are sometimes serious. 6,7 To circumvent these problems with conventional local treatments, a new therapeutic approach is needed. Tumor-specific gene therapy using tissue-specific promoter can be a rational treatment strategy for uterine cervical cancer, since the uterine cervix is readily accessible to an intratumoral approach without invasive techniques. Systemic adminis- tration of tumor-specific oncolytic adenovirus may also be an effective and safe treatment option for patients with metastatic disease. The transmembrane glycoprotein MN/CA9, which was first identified in the highly malignant human cervical carcinoma cell line HeLa, is a member of the carbonic anhydrase family with an ability to catalyze the reversible hydration of carbon dioxide to carbonic acid. 8–10 MN/ CA9 has been detected in several types of malignancies, but not in normal tissues except for epithelial cells of the gastrointestinal tract. 11–18 A high level of MN/CA9 protein expression was detected in more than 90% of uterine cervical cancer tissues. 11 These observations suggest that MN/CA9 might be a powerful biomarker for the diagnosis and treatment of uterine cervical cancer. Received December 23, 2003. Address correspondence and reprint requests to: Dr Se Joong Kim, MD, PhD, Department of Urology, Ajou University School of Medicine, San-5, Wonchon-dong, Yeongtong-gu, Suwon 442-721, Korea. E-mail: sejoong@madang.ajou.ac.kr Cancer Gene Therapy (2004) 11, 532–538 r 2004 Nature Publishing Group All rights reserved 0929-1903/04 $30.00 www.nature.com/cgt