A novel approach using transcomplementing adenoviral vectors for gene therapy of adrenocortical cancer.

Current therapies for adrenocortical carcinomas do not improve the life expectancy of patients. In this study, we tested whether a gene-transfer therapy based upon a suicide gene/prodrug system would be effective in an animal model of the disease. We employed E4- and E1A/B-depleted, herpes simplex virus-thymidine kinase-expressing adenoviral mutants that transcomplement each other within tumor cells, hereby improving transgene delivery and efficacy by viral replication in situ. Transcomplementation of vectors increased the fraction of transduced of tumor cells. This increase was accompanied by greater tumor volume reduction compared to non-transcomplementing approaches. Survival time improved with non-replicating vectors plus GCV compared to controls. However, transcomplementation/replication of vectors led to a further significant increment in anti-tumor activity and survival time (p < 0.02). In treated animals, we observed a high number of apoptotic nuclei both adjacent to and distant from injection sites and sites of viral oncolysis. Ultrastructural analyses exhibited nuclear inclusion bodies characteristic of virus production in situ, and provided further evidence that this therapy induced apoptotic cell death within tumor cells. We conclude that the efficacy of suicide gene therapy is significantly amplified by viral replication and, in combination with GCV, significantly reduces tumor burden and increases survival time.

Biosynthetic ganciclovir triphosphate: its isolation and characterization from ganciclovir-treated herpes simplex thymidine kinase-transduced murine cells.

A method is described for the preparation of ganciclovir triphosphate (GCV-TP) using murine colon cancer cells (MC38) transduced with the herpes simplex virus-thymidine kinase (MC38/HSV-tk). Murine cells transduced with viral-tk contain required viral and host enzymes needed for complete cellular synthesis of this potent antiviral metabolite. Dose response studies showed optimal intracellular levels of GCV-TP occurred after exposure of MC38/HSV-tk cells to 300 microM ganciclovir for 24 h producing 7.5 nmol GCV-TP/10(6) cells. This reflects cellular accumulation of GCV-TP to levels 25-fold greater than the medium concentration of parent drug. A simple isolation scheme included methanolic extraction and anion-exchange chromatography to recover the target triphosphate. Mass spectral analysis and selective enzyme degradation provided structural confirmation of the purified product. Biological activity of the purified GCV-TP was demonstrated by competitive inhibition experiments using human DNA polymerase alpha and HSV DNA polymerase that showed substantially greater sensitivity for the viral polymerase in agreement with previous reports. The GCV-TP obtained was further used to enzymatically prepare GCV mono- and diphosphate in high yield. This method provides an easily scalable means of preparing milligram amounts of the triphosphates of pharmacologically active acyclic nucleosides like ganciclovir.

AV.TK-mediated killing of subcutaneous tumors in situ results in effective immunization against established secondary intracranial tumor deposits.

Gene transfer vectors expressing herpes simplex thymidine kinase (HSVtk), in addition to direct killing of tumor cells, often have an associated local "bystander effect" mediated by metabolic coupling of tumor cells. A systemic antitumor effect mediated by the immune system, termed the distant bystander effect, has also been reported. We have observed the development of cytotoxic T-lymphocyte (CTL) populations and long-lasting antitumor immunity following treatment of subcutaneous tumors with an adenoviral vector expressing HSVtk (AV.TK) and ganciclovir (GCV) in rat glioma model. This vaccination effect seen with AV.TK/GCV treatment of subcutaneous tumor could even abrogate or retard growth of previously established secondary intracranial tumors.

Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection.

To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred syngeneic lymphocytes after ex vivo expansion and transduction with a chimeric receptor gene (CD4/CD3-zeta) between identical twins discordant for HIV infection. Single and multiple infusions of 10(10) genetically modified CD8(+) T cells resulted in peak fractions in the circulation of approximately 10(4) to 10(5) modified cells/10(6) mononuclear cells at 24 to 48 hours, followed by 2- to 3-log declines by 8 weeks. In an effort to provide longer high-level persistence of the transferred cells and possibly enhance anti-HIV activity, we administered a second series of infusions in which both CD4(+ )and CD8(+) T cells were engineered to express the chimeric receptor and were costimulated ex vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions of approximately 10(3) to 10(4) modified cells/10(6) total CD4(+) or CD8(+) cells persisted for at least 1 year. Assessment of in vivo trafficking of the transferred cells by lymphoid tissue biopsies revealed the presence of modified cells in proportions equivalent to or below those in the circulation. The cell infusions were well tolerated and were not associated with substantive immunologic or virologic changes. Thus, adoptive transfer of genetically modified HIV-antigen-specific T cells was safe. Sustained survival in the circulation was achieved when modified CD4(+ )and CD8(+) T cells were infused together after ex vivo costimulation, indicating the important role played by antigen-specific CD4(+) T cells in providing "help" to cytotoxic effectors. (Blood. 2000;96:467-474)

Use of a herpes thymidine kinase/neomycin phosphotransferase chimeric gene for metabolic suicide gene transfer.

Metabolic suicide gene transfer is widely applied for gene therapy of cancer, and retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene are commonly used in clinical trials. Most of these vectors contain positive selectable markers that undoubtedly facilitate the determination of viral titer and the identification of high-titer producer clones. However, the presence of additional transcriptional units may result in reduced expression of the gene of interest. The use of fusion genes expressing bifunctional proteins may help to overcome this problem. We have constructed a retroviral vector carrying the TNFUS69 chimeric gene, which originates from the fusion of the HSV-tk and neomycin phosphotransferase II genes, and evaluated the functional expression of the encoded fusion protein. In vitro, expression of the fusion gene conferred to target cells both resistance to neomycin and selective sensitivity to the antiherpetic drugs ganciclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine. Cells transduced with the fusion gene, however, showed reduced ability to phosphorylate ganciclovir compared with cells expressing the native HSV-tk. Therefore, although the fusion gene may be used as a constituent of retroviral cassettes for positive and negative selection in vitro, its usefulness for suicide gene transfer applications in vivo may depend upon the possibility of using (E)-5-(2-bromovinyl)-2'-deoxyuridine in a clinical context.

Development of a skin-based metabolic sink for phenylalanine by overexpression of phenylalanine hydroxylase and GTP cyclohydrolase in primary human keratinocytes.

Phenylketonuria, PKU, is caused by deficiency of phenylalanine hydroxylase (PAH) resulting in increased levels of phenylalanine in body fluids. PAH requires the non-protein cofactor BH4 and the rate-limiting step in the synthesis of BH4 is GTP cyclohydrolase I (GTP-CH). Here we show that overexpression of the two enzymes PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH4 supplementation. Integration of multiple PAH and GTP-CH transgenes were achieved after optimized retroviral transduction. Phenylalanine clearance was measured ex vivo in primary human keratinocytes cotransduced with PAH and GTP-CH (more than 370 nmol/24 h/106 cells), a level exceeding that of a human liver cell line (HepG2 cells). Cells overexpressing either one of the enzymes alone did not clear significant amounts of phenylalanine. Transfer of the two genes into the same cell was not necessary, since cocultivation of cells transduced separately with PAH and GTP-CH also resulted in phenylalanine clearance. Thus the experiments indicate metabolic cooperation between cells overexpressing PAH and cells overexpressing GTP-CH, possibly due to intercellular transport of synthesized BH4.

Synergy between the herpes simplex virus tk/ganciclovir prodrug suicide system and the topoisomerase I inhibitor topotecan.

An established principle of antineoplastic chemotherapy is that multidrug regimens are generally superior to single-agent therapy. This prompted us to elucidate whether the topoisomerase inhibitor topotecan (TPT) could enhance the efficacy of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system for the treatment of cancer. We assessed the interaction between these two treatments in murine MC38 and human HT-29 colon carcinoma cell lines that were genetically modified to constitutively express HSV-tk, sensitizing them to GCV. Synergistic cell killing was observed in a clonogenic assay over most of the cytotoxic dose range by the median-effect principle of Chou and Talalay (Adv. Enzyme Regul. 1984; 22:27-55). Subcutaneous tumor models, using the same cell lines in C57BL/6 and athymic nude mice, respectively, demonstrated that the combination of GCV and TPT resulted in statistically significant enhanced survival relative to single-agent treatment. In addition, nude mice bearing HT-29 tumor xenografts were treated with an Ad5 E1b Mr 55,000 attenuated replication-competent adenovirus expressing HSV-tk (Ad.TK(RC)) either alone or in combination with GCV and/or TPT. These experiments demonstrated that Ad.TK(RC) followed by GCV and TPT was more efficacious than any other treatment tested. Our results suggest that for antineoplastic therapy, molecular chemotherapy based on the HSV-tk/GCV system combined with traditional chemotherapy is a logical and practical future direction to pursue. Suicide gene therapy is the approach whereby genetically altering a cell makes it susceptible to an otherwise relatively nontoxic prodrug. By this approach it is possible to achieve relatively high concentrations of the toxic metabolites in the transduced cells while maintaining low systemic levels of the active drug. The most often used metabolic suicide gene transfer system is the HSV-tk/GCV paradigm, which is currently being used in cancer therapy or as a safety modality. The low response rate observed in the early clinical HSV-tk cancer trials may be due to failure in achieving adequate transduction efficiency and/or prodrug concentration within the tumor. The combination of such suicide gene prodrug systems with adjunctive drugs resulting in synergistic cytotoxicity might improve the clinical utility of this approach.

Enzyme prodrug gene therapy: synergistic use of the herpes simplex virus-cellular thymidine kinase/ganciclovir system and thymidylate synthase inhibitors for the treatment of colon cancer.

The goal of this study was to improve the therapeutic index of the herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) system by the addition of thymidylate synthase (TS) inhibitors. For this, we assessed the potential of GCV to synergistically interact with 5-fluorouracil (5-FU), ZD1694 (Tomudex), and (E)-5-(2-bromovinyl)-2'-deoxyuridine in HSV-tk-expressing murine MC38 STK and human HT-29 STK colon carcinoma cell lines. Synergistic cell killing was observed in a clonogenic assay over most of the cytotoxic dose range by the median-effect principle of Chou and Talalay (T. C. Chou and P. Talalay, Adv. Enzyme Regul., 22: 27-55, 1984). In a s.c. HT-29 STK xenograft tumor model, we demonstrated that the combination of GCV and 5-FU resulted in statistically significant enhanced animal survival over single-agent treatment. Furthermore, we showed that the combination of GCV and ZD1694 in association with the HSV-tk/GCV system was at least as effective as GCV/5-FU in vitro and in vivo. The mechanism for the observed synergy is most likely attributable to the increased GCV phosphorylation in the presence of the tested TS inhibitors. Our data suggest that the HSV-tk/GCV metabolic suicide gene transfer system could serve as an adjuvant of the presently used TS inhibitors, thus potentially improving the efficacy of present cancer gene therapy approaches.

Adeno-retroviral chimeric viruses as in vivo transducing agents.

Several hybrid viral gene transfer systems have been described that exploit the favorable features of the two parent viral species. We have developed a hybrid adeno-retroviral vector system to generate a retroviral vector in situ. The system consists of adenoviruses encoding MoMLV gag.pol (Axtet.gag.pol), the VSV-G viral envelope (Axtet.VSV-G), the retroviral vector LXSN expressing the neomycin phosphotransferase gene (AV-LXSN) and a transcriptional regulator to control expression of gag.pol and envelope (AV-rtTA). In vitro, biologically active retroviral vector preparations were generated following adeno-retroviral transduction of 9L rat glioma cells. In vivo the transcomplementing adeno-retroviruses were co-administered intratumorally into subcutaneous 9L glioma tumors in rats and human A375 melanoma xenografts in nude mice. In the 9L rat model, G418 cell cultures were only obtained when 9L cells were harvested from tumors injected with all four transcomplementing adeno-retroviruses. Molecular analysis of DNA extracted from 9L G418 populations derived both in vitro and in vivo showed appropriate integration of the LXSN proviral sequence. Tumor cells were harvested 1, 3 and 4 weeks after adeno-retrovirus administration to the human A375 xenografts. The percentage of G418 colonies recovered from tumors transduced with all of the transcomplementing adeno-retroviruses increased with time, whereas no increase was observed in tumors transduced with AV-LXSN alone. DNA extracted from G418 A375 cell populations showed the presence of integrated proviral sequences only in animals that received the full complement of adeno-retroviruses. These results demonstrate that adenoviral vectors expressing transcomplementing genes for retroviral proteins and retroviral vector RNAs can be used for in situ transduction of target cells.

Retrovirus-mediated WASP gene transfer corrects defective actin polymerization in B cell lines from Wiskott-Aldrich syndrome patients carrying 'null' mutations.

Boys affected with Wiskott-Aldrich syndrome (WAS) present with variable association of thrombocytopenia, eczema and immune deficiency. If untreated, WAS patients may succumb to intracerebral hemorrhages, severe infections or malignancies. Allogeneic bone marrow transplantation (BMT) can cure all aspects of the disease, but HLA-identical donors are not available to all patients and mismatched BMTs are unfortunately associated with high mortality and morbidity. The good success of HLA-matched BMT, however, makes WAS a potential candidate for hematopoietic stem cell gene therapy. WAS patients carry mutations of the Wiskott-Aldrich syndrome protein gene encoding WASP, a 502-amino acid proline-rich protein with demonstrated involvement in the organization of the actin cytoskeleton. To verify the feasibility of genetic correction for this disease, the WASP cDNA was expressed in EBV-immortalized B cell lines obtained from WAS patients using a retroviral vector. Transduced WAS cells showed levels of WASP expression similar to those found in cells from normal donors, without detectable effects on viability or growth characteristics. In addition, retrovirus-mediated expression of WASP led to improvement of cytoplasmic F-actin expression and formation of F-actin-positive microvilli, a process shown to be defective in untransduced WAS cell lines. These preliminary results indicate a potential use for retrovirus-mediated gene transfer as therapy for WAS.

Adenoviral vectors capable of replication improve the efficacy of HSVtk/GCV suicide gene therapy of cancer.

A major obstacle to the success of gene therapy strategies that directly target cancer cells is the poor vector distribution within solid tumors. To address this problem, we developed an E1b 55 kDa attenuated, replication-competent adenovirus (Ad.TKRC) which expresses the herpes simplex-1 thymidine kinase (HSVtk) gene to sensitize tumors to ganciclovir (GCV). Efficacy of this combined strategy was tested in nude mice with subcutaneous human A375 melanoma and ME180 cervical carcinomas. Intratumoral injection of a replication-defective adenoviral vector expressing HSVtk (Ad.TK) followed by GCV treatment resulted in doubling of the survival time of mice bearing A375 tumors and 20% long-term survival of mice with ME180 tumors. Treatment of tumors with Ad.TKRC without GCV resulted in a similar antitumor effect, confirming that the replicating vector has an oncolytic effect. When GCV was initiated 3 days after Ad.TKRC injection, survival of mice with each tumor type was greatly prolonged, with 60% of animals with ME180 tumors surviving for over 160 days. These results confirm that both the oncolysis caused by a replicating virus and suicide/prodrug gene therapy with HSVtk/GCV have potent antitumor effects. When combined, these two approaches are complementary resulting in a significantly improved treatment outcome.

Gene therapy for severe combined immunodeficiency caused by adenosine deaminase deficiency: improved retroviral vectors for clinical trials.

Severe combined immunodeficiency (SCID) caused by adenosine deaminase deficiency (ADA-) is the first genetic disorder to be treated with gene therapy. Since 1990 when the first trial started for 2 patients with ADA- SCID, five clinical trials enrolling 11 patients have been conducted with different clinical approaches and the results obtained from these trials have recently been reported. According to these reports, T cell-directed gene transfer was useful in the treatment of ADA- SCID whereas the retroviral-mediated gene transfer to hematopoietic stem cells was insufficient for achievement of clinical benefits. This chapter reviews several crucial problems inherent in the current retroviral technology based on the clinical data observed in these pioneering ADA gene therapy trials and presents our new retroviral vector system for the next stem cell gene therapy.

Retroviral-mediated transfer and expression of the common gamma chain into human hematopoietic progenitors.

The common gamma chain (gammac) of cytokine receptors is mutated in X-linked severe combined immunodeficiency, a lethal disorder characterized by the absence of both humoral and cellular immune defenses. Allogeneic bone marrow transplantation from HLA-identical siblings usually results in complete reconstitution of the immune system and is the current treatment of choice. Genetic correction and reinfusion of autologous hematopoietic stem cells represents an alternative therapeutic approach for those patients who lack suitable marrow donors. In this study, we show that retroviral-mediated transfer of the gammac gene results in efficient expression in CD34+ cells and high transduction rate of colony-forming progenitors.

Anti-tumor immunity generated by tumor cells engineered to express B7-1 via retroviral or adenoviral gene transfer.

We engineered B7-1 retroviral and adenoviral gene transfer systems and studied them in four immunogenic tumor models. M-MSV tumor cells, but not K-Balb, 38.2 and 205 tumor cells, when expressing B7-1 by retroviral transduction were rejected and conferred protection against a tumor challenge. Transient expression of B7-1 after transduction with adenoviruses was less efficient. We observed enhanced cytotoxic T-lymphocyte activity accompanied by increased secretion of IL-6, IFNgamma and GM-CSF. GM-CSF secretion correlated with tumor rejection. Enhanced IFNgamma but unchanged IL-4 secretion suggested a T-helper 1-mediated anti-tumor immune response.

Therapy of colon cancer with oncolytic adenovirus is enhanced by the addition of herpes simplex virus-thymidine kinase.

A major obstacle to the successful application of suicide gene therapy strategies that rely on in situ transduction of tumor cells is the poor distribution of the vector throughout the tumor mass. To address this problem, we evaluated the use of Ad.TK(RC), an E1b Mr 55,000 deleted replicating adenoviral vector engineered to express the herpes simplex virus type 1 thymidine kinase gene (HSV-tk) in combination with ganciclovir (GCV) as a treatment for human colon cancer xenografts in nude mice. We compared the efficacy of this system with that of a standard replication-deficient adenovirus expressing HSV-tk (Ad.TK) in mice bearing LS180 tumors. In this system, Ad.TK(RC) alone was as effective as a traditional Ad.TK vector in combination with GCV. The addition of GCV significantly enhanced the antitumor effect of Ad.TK(RC). Furthermore, we demonstrated that the survival of HT-29 human colon cancer xenografted mice treated with Ad.TK(RC) and GCV was prolonged compared with Ad.TK(RC) alone or with administration of a single cycle of topotecan. These data demonstrate that the addition of direct viral oncolysis to the HSV-tk/GCV suicide gene system resulted in a striking improvement in treatment efficacy and that it may offer advantages over the use of chemotherapeutic agents for treatment of localized disease.