392. p16 Cancer Gene Therapy: An Efficacious Modality in the Treatment of Nasopharyngeal Carcinoma (NPC)

392. p16 Cancer Gene Therapy: An Efficacious Modality in the Treatment of Nasopharyngeal Carcinoma (NPC)

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390. Phenotypic Knockout of Cyclin E in Breast Cancer Cells Using Novel Anti-Cyclin E sFv-Fc Fusion Intrabodies Randall W. Strube,1,2 Si-Yi Chen.2 1 Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, United States; 2Deparment of Molecular and Human Genetics, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, United States.  >                

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391. Adenovirus-Mediated Retinoblastoma 94 Gene Therapy Enhances Cisplatin-Based Chemotherapy for the Head and Neck Cancer Daqing Li, Shu-Zhen Yu, David Bray, David Van Echo, Sunil Sreedharan, Bert O′Malley.                       

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   ' "@256       Molecular Therapy Vol. 5, No. 5, May 2002, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

392. p16 Cancer Gene Therapy: An Efficacious Modality in the T reatment of Nasopharyngeal Carcinoma Treatment (NPC) Andrew Wing Cheong Lee,1 Jian-Hua Li,1 Ta-Jen Liu,3 Fei-Fei Liu.1,2 1 Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; 2Radiation Oncology, University of Toronto, Toronto, Ontario, Canada; 3MD Anderson Cancer Center, Houston, Texas, United States. B)  8B    

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CANCER, TARGETED GENE THERAPY CNE-1 cells failed to establish tumours, in contrast to adv.β-gal or control tumours. Conclusion: We have demonstrated that adv.p16, combined with irradiation, is potentially useful in NPC, where p16 expression is absent in a significant majority of patient tumours. The mechanism of cytotoxicity is likely multifactorial, including cell cycle arrest, senescence, and necrosis. Our long-term goal is to enhance the specificity and reliability of gene therapy in NPC patients, and facilitating its role as a major treatment modality of cancer therapy.

393. Infectious Delivery of the 132 kb CDKN2A / CDKN2B Complex Genomic Region Kaveh Asadi Moghaddam,1 Moksha Ranasinghe,1 E. Antonio Chiocca,1 Yoshinaga Saeki,1 Richard Wade-Martins.1 1 Molecular Neuro-Oncology Laboratories, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, United States. The chromosomal region 9p21.3 contains the CDKN2A and CDKN2B loci and is frequently altered in human cancer. The CDKN2A locus gives rise to two distinct transcripts from different promoters, encoding p16INK4a and p14ARF from overlapping reading frames. Furthermore, an additional splice variant of p16 INK4a, termed p12, is found specifically in human pancreas. Twelve kilobases upstream of the CDKN2A locus lies CDKN2B, which encodes p15 INK4b, and its alternative splice variant, p10. Another gene coding for susceptibility protein NSG-x has been reported to lie 1 kb downstream of CDKN2A. Remarkably, three of these proteins -p16INK4a, p14ARF and p15INK4b- are proven negative regulators of cell proliferation and preliminary published reports have shown that p10 and p12 also have inhibitory effects on cell growth. In total, this 42 kb region contains four promoters and seven exons which encode six different proteins. We hypothesize that delivery of the complete genomic region would offer the possibility for restoring correct cell cycle regulation to tumor cells. Gene therapy systems capable of delivery of such complex loci will also allow us to exploit the complex regulation of gene expression from genomic DNA. We tested ten different glioblastoma (Gli) cell lines for the presence of the p16INK4a, p14ARF and p15INK4b exons by PCR. Four of the ten Gli cell lines showed a deletion of all exons. In addition, we confirmed that a pancreatic cell line (PANC-1) possesses a deletion of the same region. Next, by screening the published electronic databases we obtained a fully sequenced bacterial artificial chromosome (BAC) with a 195 kb genomic DNA insert for the CDKN2A / CDKN2B region. By PCR analysis, we verified that this BAC insert carries the entire sequence of interest. We then performed preliminary transfection studies in our confirmed null cell lines and demonstrated expression of p16 INK4a from the BAC. Because of the extremely low transfection efficiency of genomic DNA constructs of >100 kb, we have developed an infectious delivery system based on Herpes simplex virus 1 (HSV-1). Amplicon vectors carrying the origin of replication (oriS) and the packaging/cleavage (pac) signal from HSV-1 offer an attractive vector system for infectious delivery of complete genomic loci because of their high transgene capacity (~150 kb), broad infectivity and high titer. We converted the BAC clone into an infectious HSV-1 amplicon vector in three stages. First, we used RecAassisted restriction endonuclease (RARE) cleavage to remove 63 kb of extreme 3′ DNA. Second, we used a Cre/loxP-mediated recombination strategy to retrofit the reduced BAC with the vectors pHG and pEHHG, creating a HSV-1 and a HSV-1/Epstein-Barr virus (EBV) hybrid amplicon, respectively. The resulting amplicon plasmids both carry the 132 kb genomic DNA insert and were designated pHSV-CDKN2A/B (147 kb) and pHSV/EBV-CDKN2A/B (154 kb). Finally, both constructs were packaged into HSV-1 virions using an improved helper virus-free system. Currently, we are confirming intact vector delivery and gene expression from the 132 kb genomic DNA insert and we will then examine the effect of gene expression from this region on tumor cell lines.

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394. Effect of Antisense bcr-abl RNA on STI571Resistant Chronic Myeloid Leukemia Colin L. Sweeney,1 Catherine M. Verfaillie,2 Junia V. Melo,3 R. Scott McIvor.1 1 Gene Therapy Program, Institute of Human Genetics, Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota, United States; 2 Stem Cell Institute, Department of Medicine, University of Minnesota, Minneapolis, Minnesota, United States; 3Department of Haematology, Imperial College School of Science, Technology and Medicine, Hammersmith Hospital, London, United Kingdom. Chronic myeloid leukemia (CML) is a disease of the hematopoietic stem cell, characterized by presence of the bcr-abl fusion oncogene. STI571 is a potent inhibitor of the p210bcr-abl tyrosine kinase, and has been very effective in inducing remission of chronic-phase CML in clinical trials. However, STI571 treatment frequently leads to relapse in patients with blast-phase CML, either due to bcr-abl gene amplification or mutations within the kinase domain of the bcr-abl oncogene. We have been testing a gene therapy approach for treatment of CML, involving marrow transduction with the LasBD retroviral vector containing a drug-resistant DHFR gene and an antisense sequence directed against the bcr-abl message. Transduction with LasBD allows for methotrexate or trimetrexate selection against cells not expressing the DHFR gene while restoring a more normal phenotype to bcr-abl+ tumor cells. The antisense component has previously been tested in STI571-sensitive bcr-abl+ 32Dp210 and MO7e-p210 cell lines, and has been found to specifically reduce bcr-abl mRNA and p210bcr-abl protein levels as well as decrease tumorigenicity of LasBDtransduced cells. STI571-resistant Baf/BCR-ABL-r1 cells with upregulated p210bcr-abl expression have been generated, and these cells as well as STI571sensitive Baf/BCR-ABL-s cells have been transduced with LasBD or LBD (a control vector containing the drug-resistant DHFR gene but lacking the bcr-abl antisense) vectors, selecting with methotrexate to obtain transduced cell populations. The effect of antisense expression is being tested in these cells to determine whether the antisense RNA can similarly reduce bcr-abl mRNA and p210 bcr-abl protein expression and decrease tumorigenicity in STI571-resistant cells. As part of a gene therapy approach, antisense expression might thus provide an effective treatment for patients with STI571-resistant CML.

395. The Development of a Novel Tricistronic Retroviral Vector for Chemoprotective Gene Therapy of Haemopoietic Stem Cells Michael D. Milsom,1 Lorna B. Woolford,1 Dorothy Gagen,1 Leslie J. Fairbairn.1 1 Gene Therapy Group, Paterson Institute for Cancer Research, Manchester, United Kingdom. The transduction of bone marrow cells with retroviral constructs containing drug resistance genes, has been shown to protect haemopoiesis from collateral damage following administration of the appropriate chemotherapeutic compounds. However, a major drawback to this strategy is the relatively poor rate of transduction within the stem cell compartment. Other authors have documented that the retroviral mediated expression of the HoxB4 homeobox protein in mice, leads to an enhanced rate and level of haemopoietic reconstitution following transplantation with gene modified bone marrow cells. Furthermore, this improved reconstitution was found to be due to a self-limiting expansion in the number of transduced stem cells. In order to combine HoxB4 activity with chemoresistance, we have created a novel tricistronic retroviral vector containing a cassette encoding the human O 6-alkyguanine-DNA alkyltransferase harbouring a point mutation which confers resistance to the tumour sensitising agent O6-benzylguanine; the foot and mouth disease virus 2a “self cleaving” moiety; human HoxB4 and finally, the enhanced green fluorescent protein driven by the internal ribosome entry site of encephalomyocarditis virus. Using this construct, we have demonstrated both alkytransferase and HoxB4 protein expression in FACs sorted green fluorescent murine fibroblasts. Moreover, we have successfully transduced primary murine bone marrow cells with this vector and are currently investigating its efficacy in vivo.

Molecular Therapy Vol. 5, No. 5, May 2002, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy