Trans-complementing adenoviral vectors for oncolytic therapy of malignant melanoma.

BACKGROUND: Despite attempts to develop efficient viral-based gene transfer therapies for the treatment of malignant tumors, only limited progress has been made to improve the efficacy of this approach. As an alternative, the use of replicating oncolytic adenoviruses with and without the expression of therapeutic transgenes is an area of active investigation. METHODS: We used a human melanoma xenograft tumor nude mouse model to test the efficacy of a bivalent vector approach consisting of two trans-complementing replication-incompetent adenoviral vectors that resulted in tumor-restricted oncolysis. We combined an E1-deleted non-replicating adenoviral vector expressing the herpes simplex virus thymidine kinase gene (AV.C2.TK) and Ad5.dl1014, an E4-deleted/E4orf4-only expressing adenovirus, to allow full replication competence when tumor cells were co-infected with both vectors. RESULTS: A375 tumors showed apoptosis at the ultrastructural level after transduction with the trans-complementing vector system that was not seen with injection of either vector alone. Apoptotic DNA fragments could be co-localized to sites of infection with the adenoviral vectors. A significant survival benefit was achieved for the trans-complementing vector treated animals compared to animals treated with either vector alone. Interestingly, the administration of GCV did not further increase animal survival over treatment with the trans-complementing system of viruses alone, and long-term survival was only seen in the trans-complementing vector treatment group. Intraperitoneal administration of a pseudo-wild-type vector Ad.dl327 resulted in significant hepatotoxicity, while intraperitoneal administration of the trans-complementing vectors resulted in only mild liver abnormalities. CONCLUSIONS: The trans-complementing vector approach using a combination of E1- and E4-deleted adenoviral vectors showed similar antitumor efficacy as reported for monovalent replicating vector systems, but may offer additional safety by reducing the risk of dissemination of the replication-competent vectors by requiring the presence of both vectors in a cell to achieve replication competence.

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.

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.

Modified human immunodeficiency virus-based lentiviral vectors display decreased sensitivity to trans-dominant Rev.

As a first step toward the development of HIV-based conditionally replicating defective interfering particles expressing trans-dominant Rev (TdRev), we studied whether mutation of the splicing signals and replacement of the RRE by the SRV-1 CTE would render these vectors less sensitive to TdRev. Vectors with mutations in the splicing signals (SD-/RRE+) yielded high titers (5 X 10(6) CFU/ml) and showed higher levels of cytoplasmic unspliced mRNA than the corresponding SD+/RRE+ vectors either in the absence of Rev, in the presence of TdRev, or in the presence of both TdRev and Rev. Proviral copies of SD-/RRE+ vectors were rescued more efficiently than SD+/RRE+ vectors when TdRev was expressed. Vectors with the SRV-1 CTE (SD+/CTE+ and SD-/CTE+) expressed high levels of cytoplasmic unspliced mRNA in the absence of Rev expression. Titers obtained with the SD-/CTE+ vectors (10(6) CFU/ml) were higher than the titers obtained with SD+/CTE+ vectors. We also tested the effect of other structural modifications such as the orientation of the expression cassette and the presence of the central polypurine tract (cPPT/CTS). We show that an expression cassette cloned in the reverse orientation with respect to the LTRs or elimination of the cPPT/CTS element severely affected vector titers. We also demonstrated that these vectors can be efficiently mobilized from their proviral state by HIV trans-complementing functions, and transduced into secondary target cells without suffering any genomic rearrangement.

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.

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.

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.

Enhancement of the herpes simplex virus thymidine kinase/ganciclovir bystander effect and its antitumor efficacy in vivo by pharmacologic manipulation of gap junctions.

Apigenin, a flavinoid, and lovastatin, an HMG-CoA reductase inhibitor, upregulated gap junction (GJ) function and dye transfer in tumors expressing GJ and were inactive in the GJ-negative tumor line N2a. N2a cells transfected with the connexin 43 gene showed restored cell-to-cell dye transfer, which could then be improved nearly fourfold by addition of apigenin. To test the drugs in HSV thymidine kinase/ganciclovir (HSV-tk/GCV) tumor killing, mixtures of 90% wild-type (WT) with 10% HSV-tk gene-modified MCA38 adenocarcinoma cells were exposed in vitro to GCV +/- apigenin or lovastatin. A significant bystander effect (BSE) was seen following GCV treatment alone, while neither apigenin or lovastatin alone had any effect on the recovery of viable tumor colonies. However, GCV-treated cultures also exposed to apigenin or lovastatin showed an increased BSE and reduced tumor cell recovery. Thirty percent of mice bearing tumors from the same mixture of 90% WT and 10% HSV-tk MCA38 cells treated with GCV alone became tumor free. Tumor-bearing mice given only two or three injections of lovastatin or apigenin during GCV treatment had a doubling of the antitumor response rate, with 60-70% of the mice achieving complete remission. These results support the hypothesis that the transfer of phosphorylated GCV from HSV-tk gene-expressing cells to neighboring WT tumor cells is a major component of the BSE and that pharmacological manipulation of GJ function with lovastatin or apigenin can result in striking improvement in the antitumor response in mice with tumors modified to contain as few as 10% HSV-tk cells.

Generation of a conditionally neo(r)-containing retroviral producer cell line: effects of neo(r) on retroviral titer and transgene expression.

We have developed a method for generating high-titer retroviral producer cell lines conditionally containing a neomycin resistance gene (neo(r)) based on the Cre/loxP system. For this, a bicistronic retroviral splicing vector carrying the green fluorescence protein (GFP) and a marker gene cassette consisting of internal ribosome entry site (IRES) and neo(r) flanked by loxP sites, was constructed and conveniently used to generate a G418 resistant vector producer cell line. Following titer determination and verification of the biological activity of the retroviral supernatants, the selectable expression cassette which was no longer required was excised from the provirus by transient Cre expression using an adenoviral vector. This strategy led to precise excision of neo(r) and generation of retroviral supernatants containing functional 'neo-less' retroviral particles without detrimental effects on the high vector titers found in the parental neo(r)-containing producer lines. GFP expression was significantly increased after the excision of neo(r), in both the producer lines and retrovirally transduced target cells. Reintroduction of neo(r) did not alter GFP expression, suggesting that the neo(r) gene and/or its gene product per se are not acting as a transcriptional silencer.

Adenovirus vectors as transcomplementing templates for the production of replication defective retroviral vectors.

We have generated an adenovirus containing a retroviral vector sequence encoding the neomycin phosphotransferase (neo) gene (AV-LXSN). AV-LXSN transduction of retroviral packaging cell lines led to production of LXSN retroviral vector with alternative viral envelopes; exposure of target cells to retroviral containing supernatants confirmed envelope specific tropism. Retroviral titers (G418 cfu/ml) were comparable to those produced by standard techniques. Retrovirus could be detected in supernatants within 24 hours of AV-LXSN transduction and persisted as long as 120 hours. Southern blot analysis of DNA purified from populations of G418 cells showed the presence of a single neo containing restriction fragment of the appropriate size that could only be generated by reverse transcription of LXSN to produce LXSN provirus. This adeno-retroviral chimeric vector system could simplify the generation and testing of different retroviral vectors, particularly where assessment of vectors with alternative envelopes carrying novel targeting ligands is required.

Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex.

We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promoter and the SV40 intron/polyadenylation site. Transgenes were prepared for cloning into this vector by introduction of compatible restriction sites by PCR. A vector expressing rat granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed using DNA-protein complex as well as by traditional recombination techniques. The efficacy of our adenoviral cloning system utilizing DNA-protein complex was two logs higher than that seen using homologous recombination. All viruses generated by directional ligation of the insert into the vector DNA-protein complexes contained the desired transgene in the correct orientation. This technique greatly simplifies and accelerates the generation of recombinant adenoviral vectors.

The bystander effect in the HSVtk/ganciclovir system and its relationship to gap junctional communication.

The bystander effect (BSE) is an interesting and important property of the herpes thymidine kinase/ganciclovir (hTK/GCV) system of gene therapy for cancer. With the BSE, not only are the hTK expressing cells killed upon ganciclovir (GCV) exposure but also neighboring wild-type tumor cells. On testing a large number of tumor cell lines in vitro, a wide range of sensitivity to bystander killing was found. Since transfer of toxic GCV metabolites from hTK-modified to wild-type tumor cells via gap junctions (GJ) seemed to be a likely mechanism of the BSE, we tested GJ function in these various tumors with a dye transfer technique and pharmacological agents known to affect GJ communication. We confirmed that mixtures of tumor cell resistant to the BSE did not show dye transfer from cell to cell while bystander-sensitive tumor cells did. Dieldrin, a drug known to decrease GJ communication, diminished dye transfer and also inhibited the BSE. Forskolin, an upregulator of cAMP did increase GJ, but directly inhibited hTK and therefore its effect on BSE could not be determined. We conclude that these observations further support port the concept that functional GJ play an important role in the BSE and further suggest that pharmacological manipulation of GJ may influence the outcome of cancer therapy with hTK/GCV.

Gut epithelial cells as targets for gene therapy of hemophilia.

Gut epithelium is an attractive target for gene therapy of hemophilia due to the large number of rapidly dividing cells that should be readily accessible to a wide range of vectors by a noninvasive route of administration. We have performed in vitro tests to determine the suitability of gut epithelial cells for gene transfer, protein synthesis, and secretion of coagulation factors VIII and IX. The results with retroviral vectors indicate that transduced epithelial cells from human, rat, or porcine small or large intestine can synthesize significant amounts of factor VIII or factor IX and that two-thirds or more of the recombinant protein is secreted in a basolateral direction (i.e., away from the lumen and toward underlying capillaries and lymphatics). Furthermore, we have demonstrated that intestinal epithelial cells are susceptible to efficient gene transfer by lipofection and adenovirus vectors. In the case of factor IX, we have produced a high-titer adenovirus vector capable of transducing gut epithelial cells resulting in synthesis of factor IX. The results of our in vitro studies indicate that gene transfer targeting gut epithelium as a new approach to hemophilia gene therapy is rational and merits in vivo studies in hemophilia animal models.

Characterization of the antitumor immune response generated by treatment of murine tumors with recombinant adenoviruses expressing HSVtk, IL-2, IL-6 or B7-1.

In a cancer gene therapy model recombinant adenoviruses expressing the herpes simplex virus thymidine kinase (HSVtk) gene were injected into tumors in situ, either alone or in combination with adenoviruses (Avs) engineered to express IL-2, IL-6 or the costimulatory molecule B7-1. HSVtk phosphorylates the prodrug ganciclovir, thus converting it into an antimetabolite which kills not only HSVtk expressing cells, but also by the 'bystander effect', neighboring untransduced tumor cells. The tumors regressed in 80% of mice upon AvTK/ganciclovir treatment: combinations with AvIL-2, AvIL-6, or AvB7-1 did not improve these results. Cured mice were protected from further challenge with wild-type tumor but not from challenges with an unrelated syngeneic tumor cell line. Since cytotoxic T lymphocyte responses in this tumor model were weak, we analyzed cytokine secretion from spleen cells of treated animals. The best correlate of antitumor immunity in this model was enhanced secretion of GM-CSF, while secretion of IL-2, IL-6 and IFN gamma was also frequently increased but not as consistently. The enhanced IFN gamma secretion associated with unchanged IL-4 secretion suggests that AvTK treatment results in a predominantly Th1-mediated antitumor immune response.

Retrovirus mediated gene transfer as therapy for adenosine deaminase (ADA) deficiency.

ADA deficiency manifests as a severe combined immunodeficiency with profound T-lymphocytopenia. Affected individuals have variable defects of both T- and B-lymphocyte function and greatly increased morbidity and mortality caused by frequent viral and bacterial infection. In 1990 a clinical protocol for the treatment of this disease using retrovirus mediated transfer of the ADA gene into peripheral lymphocytes was begun and in 1993 an amendment permitting gene transfer to CD34+ stem cells isolated from peripheral blood or from umbilical cord blood was approved. Five patients have been treated on this protocol and have been analyzed for the persistence of cells containing the transferred gene and for immunologic reconstitution.

T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years.

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA- SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

Strategies for gene therapy.

The use of retroviral-mediated gene transfer to introduce a DNA label into T cells (TIL) being used in the immunotherapy of patients with malignant melanoma finally opened the door to the clinical application of gene therapy for a wide variety of inherited and acquired diseases. The gene therapy trial for ADA deficiency SCID has demonstrated that long term stable expression of exogenous genes can be achieved in human T lymphocytes using retroviral vectors for ex vivo treatment and that significant immune reconstitution can be achieved in these patients following periodic infusions with ADA gene-corrected autologous T cells. Newer clinical applications include the insertion of genes into CD34 enriched stem cell populations, the testing of autologous tumor vaccines employing cytokine gene-modified tumor cells and the direct transfer of the herpes thymidine kinase gene into brain tumors in situ in order to render those tumors sensitive to treatment with the ordinarily non-cytotoxic drug ganciclovir.