The Morris Laboratory's efforts focus on gene therapy approaches to the treatment of cancer. Potential strategies for gene therapy of cancer include the replacement of defective tumor suppressor genes, expression of antisense oligonucleotides against oncogenes, expression of immune stimulatory molecules and cytokines, and the transfer of genes encoding enzymes that activate non-toxic drugs into cytotoxic agents. Despite initial optimism, most clinical trials have reported few responses. This is believed to be a result of the low efficiency of in vivo gene transfer achieved by current vectors. A major effort of our laboratory is the development of more effective viral vectors for gene transfer into tumors. In an effort to enhance the transduction efficiency of adenoviral-based gene transfer vectors, we generated a series of E1a-positive, E1b 55 kD-deleted and E1b 55 kD-enabled replicating adenoviral vectors expressing herpes simplex thymidine kinase (HSV-tk). These vectors are lytic and exhibit greater in vivo antitumor activity compared to a non-replicating E1-deleted HSV-tk-expressing vector. In contrast to other reports, the activity of the E1b 55 kD-deleted vectors were not restricted to cells expressing a dysfunctional p53 gene, but rather viral replication was enhanced in actively proliferating cells compared to confluent cell cultures. The E1b 55 kD-deleted vectors required administration of ganciclovir (GCV) for optimal tumor response, however, the E1b 55 kD-intact adenoviruses exhibited more robust replication and their efficacy was not improved by use of prodrug. Early (24 hours) GCV application inhibited the replication of these vectors. One of these vectors, Ad.OW34, an E1b 55 kD-intact vector resulted in significantly greater tumor regression and improved animal survival than a comparable E1b 55 kD-deleted or a non-replicating vector expressing HSV-tk. Subcutaneous administration of the E1b 55kD-intact adenovirus (Ad.OW34) to cotton rats caused no significant toxicity. Further safety studies are planned in anticipation of a clinical trial in patients suffering from advanced head and neck cancer. In a second vector enhancement approach, we engineered a novel retroviral vector for highly selective target cell gene expression using aspects of the natural life cycle of retroviruses. Moloney murine leukemia virus (MoMLV)-based vectors have been extensively used for clinical gene transfer owing to their ability to result in long-term stable gene expression in target cells. A major limitation, however, to the use of these vectors is the difficulty in generating stable retroviral vector producer cells (VPC's) capable of secreting viruses expressing toxic genes. Approaches used to overcome this problem have utilized tissue specific and inducible promoters, or complex recombination strategies. These various systems suffer from problems of inefficiency, or promoter """"""""leakiness"""""""" leading to some level of gene expression in the VPC's, or activation of the transgene in the VPC's along with the target cells after treatment with the inducer molecule. To overcome this problem, 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 contains the cDNA of interest upstream of the viral 3' LTR in reverse orientation relative to the viral transcriptional unit. Without a promoter to drive the cDNA, no gene expression is seen. An exogenous promoter is cloned in reverse orientation at the R-U5 transition of the viral 5' LTR. On transduction of target cells by the RETRACT vector, the natural life-cycle reverse transcription of the retroviral RNA copies the 5'LTR with the promoter to the 3' LTR, where it drives expression of the reverse-oriented transgene. We studied this system using green fluorescent protein (GFP) as a marker gene and several different promoters including the SV40, CMV and RSV promoters and found no significant difference in gene expression. Detailed studies of the RETRACT system used the SV40 promoter. Southern blot and PCR analysis of genomic DNA from the VPC's and target cells demonstrated the expected approximation and orientation of the SV40 promoter to the GFP transgene only in the target cells. Northern analysis of the producer and the target cells demonstrated GFP transcripts of the appropriate length only in the target cells. Expression of GFP was not detected by flow cytometry in the PG13-RETRACT VPC's, however, fluorescence was seen in the target cells transduced with RETRACT VPC supernatants. To improve gene expression several variations of the RETRACT vector were generated including a single copy (sc), double copy (dc) and self-inactivating (SIN) versions of these vectors. The GFP fluorescence intensity was the greatest in a double copy vector (RETRACT/GFPdc). These vectors were also designed with the E. coli cytosine deaminase (CD) gene in the forward orientation under the control of the retroviral 5' LTR allowing for its constitutive expression as a negative selection marker. We are currently working on generating stable RETRACT VPC's expressing the diphtheria toxin A (DT-A) to test the feasibility of this approach. The ultimate goal is to develop packaging cells lines producing retroviral vectors expressing cytotoxic genes that would continuously produce vector and yet be unaffected by the gene or their own retrovirus. These producer cells could be inoculated into a tissue or organ (i.e. a tumor or destructive rheumatoid pannus) and transduce the target tissue with the RETRACT vector over a prolonged period of time. Once the desired effect is achieved, the VPC's could be removed by treatment with 5-fluorocytosine, which is activated to a toxic metabolite (5-fluorouracil) by the CD enzyme. It is clear that most current gene therapy approaches to cancer represent local strategies that are limited by their inability to selectively target metastatic tumor. A vaccine approach currently under evaluation in clinical trials is the use of peptide pulsed dendritic cells as a method of stimulating antitumor immunity. In this approach autologous dendritic cells incubated with synthetic peptide sequences containing aminoacid mutations found in oncogenes or defective tumor suppressor genes expressed in the tumor are injected into patients in hopes of stimulating specific T cell responses against tumor. This strategy is predicated upon knowing the patient's HLA-type and that the binding affinity of a mutant peptide sequence to the MHC molecules are greater than that of the wild type protein sequence. Since joining the laboratory in May, Dr. Yoshio Sakai, M.D., Ph.D. been working on adenoviral-mediated transfer of the genes encoding these mutant proteins into dendritic cells as a cancer vaccine approach. The advantages of this approach is that it does not require the knowledge of the particular MHC class or knowing the affinity of individual peptide sequences for the MHC molecules. The mutant protein or truncated version in the case of a dominant transforming oncogene is transferred into ex vivo cultured autologous dendritic cells using high titer recombinant E1-deleted adenoviral-mediated gene transfer. The dendritic cells are then injected back into the subject where the expressed mutant gene is processed and presented by the dendritic cells in MHC. Use of an adenoviral-based vector may offer additional benefits over the synthetic peptide approach due to the strong immunostimulatory effects of the native adenoviral proteins that may act as an adjuvant. We chose to study this approach using a mutant rat HER-2/neu gene and the K-ras gene. We have generated recombinant adenoviral vectors expressing the rat HER-2/neu transmembrane and/or extracellular domains as well as several control vectors. We have been able to harvest, mature and maintain mouse bone marrow-derived dendritic cells in culture. Studies are in progress on the infection efficiency of dendritic cells using an adenovirus expressing green fluorescent protein (Ad.GFP). Preliminary data indicate that relatively high concentrations of adenovirus (MOI 100-300) are required to achieve meaningful levels of GFP expression. We are using anti-adenovirus antibody:Ad.GFP complexes to exploit the Fc-receptors that are expressed on dendritic cells as a method to improve the efficiency of the vector uptake process. Initial experiments will look at the ability to express mutant HER-2/neu in dendritic cells in vitro. Using a Balb/c mouse model we plan to study the induction of T cell responses (specific T cell proliferation and CTL) and antitumor responses against the TUBO breast cancer cell line expressing this HER-2/neu mutant gene. The ultimate goal will be to test this approach in a HER-2/neu transgenic mouse as a model. These mice developed spontaneous premalignant breast pathology that progresses to frank breast cancers beginning at 3 months of age. In collaboration with the laboratory of Dr. Jay Berzofsky, we are generating a series of E1-deleted adenoviral vectors expressing mutant K-ras oncogenes that will be used to study the effects of secondary aminoacid substitutions (epitope enhancement). We cloned wild type K-ras and two codon-12 mutants (GLYyASP and GLYyVAL) commonly found in human cancers. Using site-directed mutagenesis we will introduce additional point mutations in codons 7 and 11. These will be studied in the Berzofsky laboratory for their ability to enhance CTL responses over that achieved with the peptide approach.
| 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 |
