Gene therapy of cancer is a novel approach with the potential to selectively eradicate tumour cells, whilst sparing normal tissue from damage. In particular, gene-directed enzyme prodrug therapy (GDEPT) is based on the delivery of a gene that encodes an enzyme which is non-toxic per se, but is able to convert a prodrug into a potent cytotoxin. Several GDEPT systems have been investigated so far, demonstrating effectiveness in both tissue culture and animal models. Based on these encouraging results, phase I/II clinical trials have been performed and are still ongoing. The aim of this review is to summarise the progress made in the design and application of GDEPT strategies. The most widely used enzyme/prodrug combinations already in clinical trials (e.g., herpes simplex 1 virus thymidine kinase/ganciclovir and cytosine deaminase/5-fluorocytosine), as well as novel approaches (carboxypeptidase G2/CMDA, horseradish peroxidase/indole-3-acetic acid) are described, with a particular attention to translational research and early clinical results. J. Cell. Physiol. 187:22–35, 2001. © 2001 Wiley-Liss, Inc.

Gene directed enzyme/prodrug therapy of cancer: historical appraisal and future prospectives.

BACKGROUND

The authors have previously demonstrated nasal T-cell lymphoma (NTL) associated with Epstein–Barr virus (EBV). The detailed clinical, phenotypic, and genotypic features and the role of EBV in lymphomagenesis remain to be clarified.



METHODS

The study group consisted of 18 patients with NTL. The phenotype was determined by immunoperoxidase staining with various monoclonal antibodies. Genotypic study was done using Southern blot hybridization. The presence of EBV-encoded small nuclear early region (EBER) RNA and EBV DNA were determined by in situ hybridization. The expression of EBV-encoded nuclear antigen (EBNA) and latent membrane protein (LMP1) were identified by immunohistologic methods. Clonotypic analysis of EBV genomes was performed by Southern blot hybridization with EBV termini fragment probe.



RESULTS

The clinical features of NTL were characterized as prolonged fever (16 patients), widespread dissemination into distant sites (13 patients), and poor prognosis with a median survival of only 6 months. EBER transcripts were identified in 16 of 18 patients. Monoclonal EBV genomes EBNA1 and LMP1 were also detected in all EBER-positive cases tested. All 18 patients expressed pan-T antigens such as MT1, CD45RO, and/or CD2. The rearrangements of T-cell receptor (TCR)-β, -γ, and/or -δ genes were shown in all 11 patients tested. The natural killer (NK) cell phenotype CD56 was expressed in all EBV-positive cases tested, and was not detected in EBV-negative cases. Seven EBV-positive cases expressed a TCR-δ chain with rearranged TCR-γ or -δ genes whereas both EBV-negative cases corresponded to αβT-cell lymphoma, which expressed a TCR-β chain with a rearranged TCR-β gene.



CONCLUSIONS

These data suggest that EBV-positive NTL may be derived from the lineage of NK-like T-cells or γδT-cells, and that EBV may play a role in lymphomagenesis. Therefore, we propose that NTL which has peculiar clinical and histologic features, could be classified as a new lymphoma entity. Cancer 1996;77:2137-49.

Nasal T-cell lymphoma causally associated with Epstein-Barr virus : Clinicopathologic, phenotypic, and genotypic studies

The consistent presence of the EBV genome in certain tumors offers the potential for novel EBV-directed therapies. Switching the latent form of EBV infection present in most EBV-positive tumor cells into the cytolytic form may be clinically useful because lytic EBV infection leads to host cell destruction, and very few normal cells contain the EBV genome. It would also be therapeutically advantageous to induce expression of EBV-encoded lytic proteins that convert the nucleoside analogues ganciclovir (GCV) and 3'-azido-3'deoxythymidine (AZT) into their active, cytotoxic forms. In this report, we have explored two different approaches for activating the lytic form of EBV infection in tumors. We show that gamma-irradiation at clinically relevant doses induces lytic EBV infection in lymphoblastoid cell lines in vitro as well as in EBV-positive B-cell tumors in SCID mice. In addition, sodium butyrate (given as a single i.p. dose) is effective for activating lytic viral infection in some EBV tumor types in SCID mice. We also examined whether low-dose gamma-irradiation treatment of EBV-positive lymphoblastoid cells in vitro promotes GCV or AZT susceptibility. The combination of radiation with either GCV or AZT induced significantly more cell killing in vitro than either radiation or prodrug treatment alone. Most importantly, we found that the combination of gamma-irradiation and GCV was much more effective in treating EBV-positive lymphoblastoid tumors in SCID mice than either agent alone. Thus, GCV or AZT treatment could potentially enhance the therapeutic efficacy of radiation therapy for EBV-positive lymphomas in patients.

https://cancerres.aacrjournals.org/content/canres/60/20/5781.full.pdf

Activation of Lytic Epstein-Barr Virus (EBV) Infection by Radiation and Sodium Butyrate in Vitro and in Vivo: A Potential Method for Treating EBV-positive Malignancies

This review focuses on the prodrugs used in suicide gene therapy. These prodrugs need to satisfy a number of criteria. They must be efficient and selective substrates for the activating enzyme, and be metabolized to potent cytotoxins preferably able to kill cells at all stages of the cell cycle. Both prodrugs and their activated species should have good distributive properties, so that the resulting bystander effects can maximize the effectiveness of the therapy, since gene transduction efficiencies are generally low. A total of 42 prodrugs explored for use in suicide gene therapy with 12 different enzymes are discussed, particularly in terms of their physiocochemical properties. An important parameter in determining bystander effects generated by passive diffusion is the lipophilicity of the activated form, a property conveniently compared by diffusion coefficients (log P for nonionizable compounds and log D7 for compounds containing an ionizable centre). Many of the early antimetabolite-based prodrugs provide very polar activated forms that have limited abilities to diffuse across cell membranes, and rely on gap junctions between cells for their bystander effects. Several later studies have shown that more lipophilic, neutral compounds have superior diffusion-based bystander effects. Prodrugs of DNA alkylating agents, that are less cell cycle-specific than antimetabolites and more effective against noncycling tumor cells, appear in general to be more active prodrugs, requiring less prolonged dosing schedules to be effective. It is expected that continued studies to optimize the bystander effects and other properties of prodrugs and the activated species they generate will contribute to improvements in the effectiveness of suicide gene therapy.

/pdf/prodrugs-for-gene-directed-enzyme-prodrug-therapy-suicide-269m4rs9ev.pdf

Prodrugs for Gene-Directed Enzyme-Prodrug Therapy (Suicide Gene Therapy).

While adenovirus (Ad) gene delivery vectors are useful in many gene therapy applications, their broad tropism means that they cannot be directed to a specific target cell. There are also a number of cell types involved in human disease which are not transducible with standard Ad vectors, such as Epstein-Barr virus (EBV)-transformed B lymphocytes. Adenovirus binds to host cells via the viral fiber protein, and Ad vectors have previously been retargeted by modifying the fiber gene on the viral chromosome. This requires that the modified fiber be able to bind to the cell in which the vector is grown, which prevents truly specific vector targeting. We previously reported a gene delivery system based on a fiber gene-deleted Ad type 5 (Ad5) vector (Ad5.betagal.DeltaF) and packaging cells that express the viral fiber protein. Expression of different fibers in packaging cells will allow Ad retargeting without modifying the viral chromosome. Importantly, fiber proteins which can no longer bind to the producer cells can also be used. Using this approach, we generated for the first time pseudotyped Ad5.betagal.DeltaF particles containing either the wild-type Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the Ad3 fiber. Particles equipped with the chimeric fiber bound to the Ad3 receptor rather than the coxsackievirus-adenovirus receptor protein used by Ad5. EBV-transformed B lymphocytes were infected efficiently by the Ad3-pseudotyped particles but poorly by virus containing the Ad5 fiber protein. The strategy described here represents a broadly applicable method for targeting gene delivery to specific cell types.

Adenovirus Vector Pseudotyping in Fiber-Expressing Cell Lines: Improved Transduction of Epstein-Barr Virus-Transformed B Cells

Epstein-Barr virus (EBV)-positive lymphomas are frequent among immunosuppressed patients. We have examined the feasibility of killing EBV-immortalized B lymphocytes by gene transfer involving the use of "suicide" genes whose expression in target cells renders them susceptible to killing by a prodrug. We examined two gene/prodrug pairs: the Escherichia coli cytosine deaminase (CD) gene with the prodrug 5-fluorocytosine (5-FC), and the herpes simplex virus thymidine kinase (HSV-TK) gene with the prodrug ganciclovir. Retroviral vectors and drug selection were used to obtain CD or HSV-TK expression in cells. Both the CD/5-FC and the HSV-TK/ganciclovir combinations yielded substantial killing of EBV-immortalized B lymphocytes in vitro, although the CD/5-FC regimen had a significantly greater therapeutic margin than the HSV-TK/ganciclovir combination. The CD/5-FC pair, but not the HSV-TK/ganciclovir pair, was shown to have a "bystander killing effect" in vitro. When only 30% of the cells expressed the suicide gene, scid mouse tumors regressed in both the CD/5-FC regimen and the HSV-TK/ganciclovir regimen, documenting an in vivo bystander effect with both regimens. However, a greater percentage of tumors completely regressed with the CD/5-FC regimen. Overall, the sum of our data indicates that the CD/5-FC combination is the more promising regimen for treatment of EBV-associated lymphomas in vivo.

Killing Epstein-Barr virus-positive B lymphocytes by gene therapy: comparing the efficacy of cytosine deaminase and herpes simplex virus thymidine kinase

Publisher Summary Epstein-Barr virus (EBV) is a herpesvirus that infects all human populations. It is carried in “latent” form in peripheral blood B lymphocytes and is shed in the form of virus particles from a site in the oropharynx. This chapter discusses the expression and function of EBV genes during latency. EBV transforms the B lymphocytes of humans and other primates to continuous proliferation in culture, a process called immortalization. The resultant, latently infected lymphoblastoid cell lines (LCLs), support a predominantly latent viral life cycle in which only a few of the 100 or so viral genes are expressed. Viral genes expressed during latency are candidate immortalizing genes. Of all the viral gene products of the latent cycle, function has been demonstrated most clearly for Epstein-Barr nuclear Antigen 1 (EBNAl). The chapter discusses the structure of viral transcripts and promoters for the EBV latent genes and perspective on viral gene expression in the LCL model. The discovery that EBV in BL tumors expresses only EBNAl clearly limits the applicability of the LCL model to other situations. RNA from BL biopsy material and from group I BL cell lines is clearly the focus of study. Information from such studies, particularly the structure of EBNA 1 -expressing transcripts, could aid polymerase chain reaction studies of viral gene expression in peripheral blood B lymphocytes from healthy seropositive individuals.

Epstein-Barr virus in B lymphocytes: viral gene expression and function in latency.

Bidirectional Transcription oftheEpstein-Barr VirusMajor Internal Repeat

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Killing Epstein-Barr virus-positive B lymphocytes by gene therapy: comparing the efficacy of cytosine deaminase and herpes simplex virus thymidine kinase

Epstein-Barr virus in B lymphocytes: viral gene expression and function in latency.

Bidirectional Transcription oftheEpstein-Barr VirusMajor Internal Repeat