TISSUE TARGETED GENE EXPRESSION AND ANIMAL MODELS 1372. An Oncolytic Adenovirus for the Treatment of Nasopharyngeal Carcinoma (NPC) Marie C. Chia,1 Jian-Hua Li,1 Craig Strathdee,3 Dolly Huang,4 Henry J. Klamut,1 Fei-Fei Liu.1,2 1 Medical Biophysics, Ontario Cancer Institute/Princess Margaret Hospital/University of Toronto, Toronto, Ontario, Canada; 2 Radiation Oncology, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Ontario, Canada; 3Immunex Corporation, Seattle, Washington, United States; 4Chinese University of Hong Kong, Hong Kong, China. Rationale: Success for adenoviral cancer gene therapy has been limited by lack of tumor-specific cytotoxicity and efficient distribution of the vector within the tumor mass. We have recently succeeded in regulating gene expression in nasopharyngeal carcinoma (NPC) by exploiting the exclusive presence of the Epstein Barr virus (EBV) genome in the cancer cells. When the latently expressed EBV-gene product, EBNA-1, binds to a family of repeats (FR) sequence, located in the oriP of the EBV genome (oriP-FR), this results in transcriptional activation of downstream genes. We therefore modified this promoter by juxtaposing the FR sequence to a minimal CMV promoter, and demonstrated 1000-fold induction of gene expression in an EBV-positive NPC system (Li J-H et al. Cancer Res. 2002;62:171.). This transcriptional targeting strategy now allows us to address the biodistribution challenge through the construction of a conditionally replicating adenovirus under the control of the oriP-FR promoter. Methods and Materials: The E1A transcriptional unit required for adenoviral replication was cloned downstream of the EBV-responsive, oriP-FR promoter sequence, resulting in an novel oriP.E1A shuttle plasmid. Through recombination with pJM17, a novel adenovirus adv.oriP.E1A was then generated. E1A and adenoviral fiber knob protein expression was determined using Western blot analysis, cytotoxicity was evaluated in EBV-positive NPC C666-1 cells and a variety of other cancer cell lines using the MTT assay. Results: Western blotting for E1A demonstrates a time-dependent increase in expression in the C666-1 cells, in contrast to minimal expression in the EBV-negative CNE-1 and CNE-2Z NPC cells. The addition of 6 Gy appeared to increase E1A expression. Extensive cell death was observed in the C666-1 cells 3 days after infection with 5 or 10 pfu/cell of adv.oriP.E1A. In contrast, no evidence of cytotoxicity was observed in a panel of other human cell lines including NPC fibroblasts (KS-1) and several EBV-negative malignant cell lines (A549, Saos-2, CNE-1). The MTT assay corroborates a dose-dependent cytotoxic effect of adv.oriP.E1A in the C666-1 cells. Western blotting for adenoviral fiber knob protein demonstrated a time-dependent increase in expression, indicating viral replication. The effects of adv.oriP.E1A on tumorigenicity and potential therapeutic benefit in vivo are currently being evaluated. Conclusion: We have successfully constructed a novel selectively oncolytic virus for EBV-positive NPC cells, demonstrating significant cytotoxicity. This therapeutic strategy should now allow us to successfully address the in vivo transduction challenge of cancer gene therapy.
1373. Gene Therapy Cures Male Mice Infertility: Restoration of Spermatogenesis in Azoospermic Sl/Sld Mice by Lentiviral Gene Transfer Masahito Ikawa,1 Vinay Tergaonker,1 Narumi Ogonuki,2 Kimiko Inoue,2 Atsuo Ogura,2 Inder Verma.1 1 Laboratory of Genetics, The Salk Institute, San Diego, California, United States; 2Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, Japan.
weeks of age and lacZ expression was analyzed two months after injection. Sertoli cells were easily transduced with either adeno- or lenti- viral vectors and the lacZ expression was observed in approximately 70% of testicular tubules. However, adeno-viral vector showed considerable toxicity. The 390 offspring obtained from 5 males treated with lentiviral vector was subjected to PCR analysis and none of them showed evidence of germline transmission of transgene. When the lentiviral vector carrying KL2 gene under the control of CMV promoter was injected into Sl/Sld mutant, the restoration of spermatogenesis was observed in all the recipient testes (n=11). Approximately 60% of tubules in the treated testis contained differentiating spermatocytes (0% in Sl/Sld mutant and 100% in wildtype). The sperm collected from recipient testis were able to generate normal pups by in vitro fertilization. No-germ line transmission of transgene was confirmed in all of the 13 pups. The efficient gene transfer by lentiviral vector in Sertoli cells offers the possibility of gene therapy for male infertility.
1374. Treatment of β-Thalassemia in the Mouse by Regulated Expression of AAV Encoded Erythropoietin Julie C. Johnston,2 Victor M. Rivera,1 Guang-ping Gao,2 John Tazelaar,2 A. David Moscioni,2 Tim Clackson,1 James M. Wilson.2 1 ARIAD Pharmaceuticals, Inc., Cambridge, MA, United States; 2 Institute for Human Gene Therapy, Philadelphia, PA, United States. Application of gene therapy for the delivery of secreted therapeutic proteins has great potential. Most applications, however, will require precise control over the expression of the transgene to assure therapeutic levels without toxicity. We have developed a system for regulating the transcription of a gene through the dimerization of engineered transcription factors with a small molecule drug based on rapamycin. This system was evaluated in the treatment of a murine model of βthalassemia, and compared to an unregulated (constitutive) system. Mice heterozygous for deletions of both b1 (β major) and b2 (β minor) adult globin chains were used as a model for thalassemia intermedia. For unregulated expression, animals were injected intramuscularly with AAV vectors expressing murine erythropoietin (Epo) from a constitutive promoter [i.e., immediate early gene from cytomegalovirus (CMV)]. Administration of 10E11 of AAV-CMVEpo corrected the anemia and abnormal red blood cell morphology found in the β-thalassemic mice. However, within eight weeks, serum Epo increased to very high levels leading to severe polycythemia and eventually death in all treated animals. To achieve regulated expression, β-thalassemic mice also were injected with AAV vectors expressing inducible Epo and the chimeric transcription factors. Subsequent induction with rapamycin led to a dose dependent increase in Epo and normalization of hematocrit and red blood cell morphology, but without the development of polycythemia. There was no detectable expression of Epo in the absence of rapamycin and induction was reversible. These studies illustrate the importance of regulating the expression of biologically active secreted proteins when delivered via stably engrafted genes. This strategy could be considered in the treatment of Epo responsive anemias. Victor M. Rivera and Tim Clackson hold equity in ARIAD Pharmaceuticals James M. Wilson holds equity in Targeted Genetics, Inc.
Today, up to 20% of couples are infertile in worldwide and 30-50% of them is attributable to male factor infertility. Approximately 70-90% of male infertility is caused by disrupted or impaired spermatogenesis with clinical aspect of azoo- or oligospermia. Disruption of spermatogenesis is thought to be of primarily genetic origin. The Sl/Sld mutant mice offer a model system in which lack of transmembrane type c-kit ligand (KL2) expression on somatic Sertoli cell surface results in disruption of spermatogenesis. We have investigated the transduction performance of adeno-, retro-, adeno associated-, and lenti- viral vectors in testicular cells. Viral vectors were designed to carry lacZ reporter gene under the control of CMV promoter. B6D2F1 wild-type males were injected with viral vectors at six
Molecular Therapy Vol. 5, No. 5, May 2002, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy