The need to develop new approaches for cancer therapy led us to test a new strategy for gene delivery using SV40 pseudovirions carrying a lethal gene. The use of Pseudomonas exotoxin A (PE) to kill cancer cells was demonstrated successfully by Pastan and colleagues.1
Pseudomonas Exotoxin A is a 66-kDa pathogenic protein secreted by Pseudomonas aeruginosa
as a proenzyme, under the selective pressure of a low iron environment. This toxin assists bacteria in the invasion of animal tissues, including human tissues. As such, it enters cells via the low-density lipoprotein receptor-related protein (LRP). Within the cell, PE is cleaved into two peptide fragments linked by a disulfide bond. When this bond is broken, the enzymatically active C-terminal fragment translocates to the cytosol, where it inactivates elongation factor 2, thereby preventing protein synthesis and promoting apoptotic cell death.2,3
PE is comprised of three major domains: domain Ia is the cell binding domain and domain Ib may serve to enhance toxin stability; domain II harbors the protease processing site and mediates translocation; and domain III has ADP-ribosylating activity, and therefore is responsible for the toxic activity of the protein.2
Pastan, Fitzgerald and colleagues created recombinant immunotoxins (RIT) in E.coli.1
They fused the Fv portion of a monoclonal antibody (MAb) in a single chain form directly to mutants of PE missing domain 1a. The advantage of this method is that it combines the higher specificity of the antibody with the killing power of the toxin. In order to achieve stability of the RIT at 37°C, Brinkman and colleagues4,5
designed a new molecule in which the light and heavy chains of the Fv portion are held together by a disulfide bond. Trials of RIT have shown that one of them, BL22, can produce complete remissions in many patients with drug resistant leukemia.6
Suicide gene therapy is one of several strategies7
used to deliver genes to cancer cells by converting non-toxic prodrugs into active chemotherapeutic agents capable of killing cancer cells. This approach enables selective killing of cells; a maximal therapeutic effect is achieved while systemic toxicity is limited.8
Despite the so-called “bystander effect”, studies show that the efficacy of suicide gene therapy of tumors is limited by the efficiency of delivery of the vector to the tumor and the less than 100% efficiency of killing by the activated prodrug.9
However, some have found that it is possible to improve cytotoxicity by combining suicide gene therapy with conventional chemotherapy. There are certain drawbacks that need to be taken into account when dealing with viral gene delivery systems, such as the bloodstream's rapid clearance of the virus-based gene transfer system. Consequently, the development of synthetic gene delivery vectors (non-viral gene delivery systems) is required10
in order to achieve the goal of delivering the suicide gene to its target.
Simian virus 40 (SV40) is an attractive potential vector for gene transfer to kill cancer cells. The vectors are prepared with nuclear extract of SF9 insect cells containing the main viral capsid protein of the SV40 wild-type virus, VP1. For the transfer of circular DNA encapcidated in VP1, the SV40 major capsid protein demonstrates high transduction efficiency and can be introduced into a wide variety of human, murine and monkey tissues. In vitro
packaging of DNA with SV40 capsid protein enables efficient delivery of plasmids with a length of up to 17.7 kb. Moreover, it does not require any SV40 sequence, thus providing efficient gene delivery at the same level of safety when using nonviral vectors.11-13
In this study, we demonstrate delivery of PE38 toxin with or without a combination of the chemotherapeutic agent doxorubicin. SV40 pseudovirion delivery of PE was found to be effective in the treatment of human adenocarcinomas growing in mice either by direct injection or systemically. Using a combined treatment of PE38 with doxorubicin, we were able to reduce the side effects of chemotherapy.