My laboratory's efforts focus on gene transfer for the treatment of cancer. Gene therapy strategies for cancer treatment include the replacement of defective tumor suppressor genes, expression of antisense oligonucleotides to inhibit the effects of transforming oncogenes, expression of immune stimulatory molecules and cytokines in tumors, and the transfer of genes encoding enzymes that locally activate non-toxic drugs into cytotoxic agents in the tumor. Despite initial optimism, patient trials using a number of these strategies have reported few clinical responses. A major reason for this is the low efficiency of gene transfer achieved by most currently used vectors. Even replicating viruses as vectors have shown limited efficacy. My laboratory has focused on the development of more effective viral gene transfer vectors and the study of antitumor vaccination by gene transfer. With respect to the first area, we engineered a unique retroviral vector for highly selective gene expression by exploiting the natural life cycle of retroviruses. Moloney murine leukemia virus (MoMLV)-based vectors have been used extensively for clinical gene transfer owing to the fact that they can achieve long-term gene expression. One major limitation of these vectors is the difficulty in generating stable retroviral vector producer cells (VPC's) secreting viruses that express directly cytotoxic genes. Efforts by others to overcome this problem have used tissue-specific or inducible promoters, or complex viral recombination strategies. These systems suffer from the problems of inefficiency, promoter """"""""leakiness"""""""" leading to some level of gene expression in the VPC's, and co-activation of the transgene in the VPC's after exposure to the inducer molecule. To overcome these, we generated a series of MoMLV-based retroviral vectors encoding a REverse Transcription-ACtivated Transgene (RETRACT) that restricts gene expression to target cells. The prototypical RETRACT vector has the cDNA of interest inserted upstream of the viral 3' LTR in reverse orientation relative to the viral transcriptional unit. Without a promoter to drive the cDNA, there is no gene expression. An exogenous promoter is cloned in reverse orientation at the R-U5 transition of the viral 5' LTR. On transduction of the target, reverse transcription of the retroviral RNA copies the 5'LTR along with the promoter to the 3' LTR, where it then drives expression of the reverse-oriented transgene. We tested this system using green fluorescent protein (GFP) as a reporter gene using several different promoters including the SV40, CMV and RSV promoters. Molecular analysis of genomic DNA from the VPC's and target cells demonstrated the expected approximation and orientation of the promoter to the transgene only in the target cells. Northern analysis of the producer and the target cells demonstrated GFP transcripts of the appropriate size only in the target cells. GFP expression was not detected in the RETRACT VPC's; however, fluorescence was seen in the target cells transduced with RETRACT VPC supernatants. To improve gene expression several modifications of the RETRACT vector were generated including a single copy (sc), double copy (dc) and self-inactivating (SIN) versions. GFP fluorescence intensity was the greatest in a double copy vector. We are currently testing this system using Diphtheria A toxin and Pseudomonas exotoxin as a """"""""proof of principle"""""""" and a manuscript is in its final stages of preparation. Our second area of interest is the use of gene altered dendritic cells as an approach to antitumor vaccination. Most """"""""gene therapy"""""""" of cancer represents local strategies that are limited in their ability to treat remote sites of disease or target metastatic tumor. Currently in clinical trials in the Metabolism Branch is a vaccination strategy using tumor epitope-pulsed dendritic cells (DC's) to stimulate specific antitumor immunity. In this approach autologous dendritic cells incubated with synthetic peptide sequences of mutations found in oncogenes or tumor suppressor genes that are expressed in the tumor. Patients' are then vaccinated with these modified DC's in hopes of stimulating specific T cell responses against their tumor. This strategy is predicated upon knowing the patient's MHC-type and that the binding affinity of the peptide sequence to a specific MHC molecule is greater than that of the wild type peptide sequence. In the laboratory we have been exploring an alternative approach involving transfer of the gene for the tumor antigen into DC's. This approach offers several advantages: (1) the tumor antigen can be expressed by gene transfer without a detailed knowledge of the epitope or its binding affinity for a particular MHC molecule; (2) the DC's may naturally process the antigen leading to more effective presentation of epitopes; (3) more epitopes against which an immune response may be directed are found in the intact antigen than a short peptide sequence; (4) gene expression provides a """"""""continuous"""""""" source of antigen that may overcome problems of low affinity epitopes, and viral proteins associated with the vector may stimulate DC maturation and enhance the ability to generate immune responses. Dr. Yoshio Sakai and Brian Morrison in the laboratory have been working on adenoviral-mediated transfer of genes encoding tumor antigens into DC's where the expressed antigen is processed and presented by the DC's in the context of MHC to interact with T cells. The DC's are then used to vaccinate our animal tumor models. We are studying this approach using HER-2/neu and the K-ras oncogenes as targets. Dr. Sakai generated a series of recombinant adenoviral vectors encoding the HER-2/neu extracellular and transmembrane domains (Ad.HER-2.ECDtm), extracellular only (Ad.HER-2.ECD), and a control vector expressing no transgene (Ad.null). We studied effectiveness of recombinant adenoviruses in transferring reporter genes into mouse bone marrow-derived dendritic cells. We found that murine DC's could be efficiently transduced with an adenovirus expressing green fluorescent protein (Ad.GFP) at relatively low vector concentrations. Infection of immature DC's with adenoviruses induced the maturation and co-culture with transduced DC's stimulated greater proliferation of splenocytes in mixed lymphocyte culture compared to uninfected DC's. We are studying vaccination using dendritic cells transduced with these vectors in a transgenic mouse model of HER-2/neu induced breast cancer. BALB-neuT mice transgenic for the rat HER-2/neu oncogene spontaneously develop breast cancers beginning at 14 to 15 weeks of age. Using this approach over 2/3 of the mice remained tumor free at 28-weeks of age compared to none of the mice in the groups that were treated with DC's alone or with DC's transduced with Ad.null. The protective effect of the vaccination was not affected by pre-existing immunity to adenovirus. Somewhat surprisingly, antibody depletion studies indicated that CD4+ and not CD8+ T cells were critical for the vaccines effectiveness. This work has been submitted for publication and was the subject of talks at both the American Society for Gene Therapy and the International Society for Biological Therapy in 2003. Studies continue in this model looking at ways to block the activity of CD4+ T regulatory cells that may function to inhibit the immune response in older animals and those with larger tumor burdens. Brian Morrison has been working at trying to translate this work to human cells. He has been studying the effect and efficiency of adenoviral-mediated gene transfer into human peripheral blood monocyte-derived dendritic cells.
| Park, Jong Myun; Terabe, Masaki; Sakai, Yoshio et al. (2005) Early role of CD4+ Th1 cells and antibodies in HER-2 adenovirus vaccine protection against autochthonous mammary carcinomas. J Immunol 174:4228-36 |
| Wolkersdorfer, G W; Morris, J C; Ehninger, G et al. (2004) Trans-complementing adenoviral vectors for oncolytic therapy of malignant melanoma. J Gene Med 6:652-62 |
| Kobayashi, Hisataka; Kawamoto, Satomi; Sakai, Yoshio et al. (2004) Lymphatic drainage imaging of breast cancer in mice by micro-magnetic resonance lymphangiography using a nano-size paramagnetic contrast agent. J Natl Cancer Inst 96:703-8 |
| Berzofsky, Jay A; Terabe, Masaki; Oh, SangKon et al. (2004) Progress on new vaccine strategies for the immunotherapy and prevention of cancer. J Clin Invest 113:1515-25 |
| Morris, John C (2003) Cancer gene therapy: lessons learned from experiences with chemotherapy. Mol Ther 7:717-9 |
