Gene Therapy for Prostate Cancer Using Bacterial MazF Suicide System

PUBLIC ABSTRACT

Over the past decade, significant progress has been made in our ability to diagnose and treat patients with prostate cancer. Despite this encouraging survival trend, prostate cancer remains the second most common cause of cancer death among men. Gene therapy represents a powerful new alternative for cancer treatment. Several prototypes of gene therapy protocols have been attempted in preclinical studies for the treatment of prostate cancer, including the delivery of suicide genes encoding proapoptotic proteins or causing the activation of a prodrug that induces selective cytoxicity and destruction of tumor cells. Among the promising new therapeutic agents are several ribonucleases that have been included in a specific group of enzymes that exhibit various biological activities in addition to their catalytic action.

We recently discovered that a suicide gene (mazF) from E. coli functions as an mRNA interferase that cleaves all cellular mRNAs in a sequence (ACA)-specific manner. Importantly, this enzyme is a part of a unique genetic system consisting of a pair of genes encoding two components, a stable toxin (MazF) and an unstable antitoxin (MazE). We also found that MazF introduction in mammalian cells causes very effective programmed cell death, which can be prevented by MazE expression. Furthermore, we found that solid tumors created in nude mice by injecting human cancer cells were dramatically regressed by inducing the mazF gene by oral administration of tetracycline. The human cancer cells injected contained the mazF gene that was engineered to be inducible by tetracycline. This result indicates that the gene for the mRNA interferase has an excellent potential for use for gene therapy.

In the present proposal we will attempt to create a new tissue-specific cytotoxic recombinant adeno-associated virus (AAV), with which we will deliver the mazF gene to prostate cancer cells to induce their death. We have chosen AAV as a vector because data accumulated so far from a variety of preclinical and clinical trials indicate that AAV-based therapeutics have excellent safety properties and can provide long-term expression in a variety of tissues including prostate cancer cells.

The first specific aim is to engineer a recombinant virus carrying the mazF suicide gene so that the suicide gene can be induced only in prostate cancer cells leading to cell death. We will also engineer a cell line in which the recombinant virus particles are able to assemble in a very high titer. This can be achieved by introducing the antitoxin gene (mazE) in the cell line so that MazF toxicity upon virus assembly can be obviated. The preparation of high titer virus is one of the most critical factors for successful therapeutic application of the recombinant virus. The second specific aim is to evaluate the cytotoxic effects of MazF delivered by recombinant viruses with different regulatory elements in several human cancer cell lines in order to screen the most effective viral construct.

The third specific aim is to evaluate the effectiveness of the recombinant virus in inhibiting tumor cell growth in tumor xenograft and orthotopic mouse models. Xenografts, and especially orthotopic tumors, closely resemble the situation in the human patients, maintaining the interaction with the prostate environment. We will test the efficiency of recombinant virus toxicity against solid tumors derived from human cancer cells in immuno-deficient mice. We will also evaluate the therapeutic effectiveness of the combination of the virus-mediated gene therapy with chemotherapy and radiotherapy. We anticipate that this study will provide a novel effective therapeutic strategy for advanced prostate cancer and will be a powerful weapon in the battle against this devastating disease.