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Improving the chances of curing patients with cancer who have had surgery to remove metastatic sites of disease is a priority area for cancer research. Pexa-Vec (Pexastimogene Devacirepvec; JX-594, TG6006) is a principally immunotherapeutic oncolytic virus that has reached late-phase clinical trials. We report the results of a single-center, nonrandomized biological end point study (trial registration: EudraCT number 2012-000704-15), which builds on the success of the presurgical intravenous delivery of oncolytic viruses to tumors. Nine patients with either colorectal cancer liver metastases or metastatic melanoma were treated with a single intravenous infusion of Pexa-Vec ahead of planned surgical resection of the metastases. Grade 3 and 4 Pexa-Vec–associated side effects were lymphopaenia and neutropaenia. Pexa-Vec was peripherally carried in plasma and was not associated with peripheral blood mononuclear cells. Upon surgical resection, Pexa-Vec was found in the majority of analyzed tumors. Pexa-Vec therapy associated with IFNα secretion, chemokine induction, and resulted in transient innate and long-lived adaptive anticancer immunity. In the 2 patients with significant and complete tumor necrosis, a reduction in the peripheral T-cell receptor diversity was observed at the time of surgery. These results support the development of presurgical oncolytic vaccinia virus-based therapies to stimulate anticancer immunity and increase the chances to cure patients with cancer.

Supplementary Figure from Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Anticancer Immunity in Patients

Supplementary Data from Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Anticancer Immunity in Patients

Improving the chances of curing patients with cancer who have had surgery to remove metastatic sites of disease is a priority area for cancer research. Pexa-Vec (Pexastimogene Devacirepvec; JX-594, TG6006) is a principally immunotherapeutic oncolytic virus that has reached late-phase clinical trials. We report the results of a single-center, nonrandomized biological end point study (trial registration: EudraCT number 2012-000704-15), which builds on the success of the presurgical intravenous delivery of oncolytic viruses to tumors. Nine patients with either colorectal cancer liver metastases or metastatic melanoma were treated with a single intravenous infusion of Pexa-Vec ahead of planned surgical resection of the metastases. Grade 3 and 4 Pexa-Vec–associated side effects were lymphopaenia and neutropaenia. Pexa-Vec was peripherally carried in plasma and was not associated with peripheral blood mononuclear cells. Upon surgical resection, Pexa-Vec was found in the majority of analyzed tumors. Pexa-Vec therapy associated with IFNα secretion, chemokine induction, and resulted in transient innate and long-lived adaptive anticancer immunity. In the 2 patients with significant and complete tumor necrosis, a reduction in the peripheral T-cell receptor diversity was observed at the time of surgery. These results support the development of presurgical oncolytic vaccinia virus-based therapies to stimulate anticancer immunity and increase the chances to cure patients with cancer.

Purpose: Transfer of prodrug activation systems into tumors by using replication-deficient viruses has been suggested to be an effective method for achieving high local and low systemic anticancer drug concentrations. However, most current suicide gene therapy strategies are still hindered by poor efficiency of in vivo gene transfer, inefficient tumor penetration, limited bystander cell killing effect, and need of large prodrug doses. We hypothesized that local amplification provided by a replication competent platform would help overcome these limitations.Experimental Design: We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Δ24FCU1 expressing the fusion suicide gene FCU1. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil.Results: We examined the efficacy of Ad5/3-Δ24FCU1 and the replication-defective control Ad5/3-FCU1 with and without 5-FC. FCU1 expression was confirmed by Western blot, whereas enzymatic conversion levels in vitro and in vivo were determined by high-performance liquid chromatography separation. Significant antitumor effect was observed in vitro and in vivo in a murine model of head and neck squamous cell carcinoma. Although we observed a decrease in viral DNA copy number in vitro and in tumors treated with Ad5/3-Δ24FCU1 and 5-FC, suggesting an effect on virus replication, the highest antitumor effect was observed for this combination.Conclusions: It seems feasible and efficacious to combine adenovirus replication to the FCU1 prodrug activation system. Clin Cancer Res; 16(9); 2540–9. ©2010 AACR.

Supplementary Data from Targeted Chemotherapy for Head and Neck Cancer with a Chimeric Oncolytic Adenovirus Coding for Bifunctional Suicide Protein FCU1

Pexa-Vec is an engineered Wyeth-strain vaccinia oncolytic virus (OV), which has been tested extensively in clinical trials, demonstrating enhanced cytotoxic T cell infiltration into tumours following treatment. Favourable immune consequences to Pexa-Vec include the induction of an interferon (IFN) response, followed by inflammatory cytokine/chemokine secretion. This promotes tumour immune infiltration, innate and adaptive immune cell activation and T cell priming, culminating in targeted tumour cell killing, i.e., an immunologically ‘cold’ tumour microenvironment is transformed into a ‘hot’ tumour. However, as with all immunotherapies, not all patients respond in a uniformly favourable manner. Our study herein, shows a differential immune response by patients to intravenous Pexa-Vec therapy, whereby some patients responded to the virus in a typical and expected manner, demonstrating a significant IFN induction and subsequent peripheral immune activation. However, other patients experienced a markedly subdued immune response and appeared to exhibit an exhausted phenotype at baseline, characterised by higher baseline immune checkpoint expression and regulatory T cell (Treg) levels. This differential baseline immunological profile accurately predicted the subsequent response to Pexa-Vec and may, therefore, enable the development of predictive biomarkers for Pexa-Vec and OV therapies more widely. If confirmed in larger clinical trials, these immunological biomarkers may enable a personalised approach, whereby patients with an exhausted baseline immune profile are treated with immune checkpoint blockade, with the aim of reversing immune exhaustion, prior to or alongside OV therapy.

Oncolytic virus (OV) therapy has emerged as a promising approach for cancer treatment with the potential to be less toxic and more efficient than classic cancer therapies. Various types of OVs in clinical development, including Vaccinia virus (VACV)-derived OVs, have shown good safety profiles, but limited therapeutic efficacy as monotherapy in some cancer models. Many different methods have been employed to improve the oncolytic potency of OVs. In this study, we used a directed evolution process, pooling different strains of VACV, including Copenhagen, Western Reserve and Wyeth strains and the attenuated modified vaccinia virus Ankara (MVA), to generate a new recombinant poxvirus with increased oncolytic properties. Through selective pressure, a chimeric VACV, deVV5, with increased cancer cell killing capacity and tumor selectivity in vitro was derived. The chimeric viral genome contains sequences of all parental strains. To further improve the tumor selectivity and anti-tumor activity of deVV5, we generated a thymidine kinase (TK)-deleted chimeric virus armed with the suicide gene FCU1. This TK-deleted virus, deVV5-fcu1 replicated efficiently in human tumor cells, and was notably attenuated in normal primary cells. These studies demonstrate the potential of directed evolution as an efficient way to generate recombinant poxviruses with increased oncolytic potency, and with high therapeutic index to improve cancer therapy.

B Heinrich,1 J Klein,1 M Delic,1 K Goepfert,1 V Engel,1 L Geberzahn,1 M Lusky,2 P Erbs,2 X Preville,3 M Moehler1 1First Department of Internal Medicine, University Medical Center Mainz, Mainz, Germany; 2Transgene SA, Illkirch-Graffenstaden, 3Amoneta Diagnostics, Huningue, France Abstract: Oncolytic virotherapy is an emerging immunotherapeutic modality for cancer treatment. Oncolytic viruses with genetic modifications can further enhance the oncolytic effects on tumor cells and stimulate antitumor immunity. The oncolytic vaccinia viruses JX-594-GFP+/hGM-CSF (JX-GFP) and TG6002 are genetically modified by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF) or transforming 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). We compared their properties to kill tumor cells and induce an immunogenic type of cell death in a human melanoma cell model using SK29-MEL melanoma cells. Their influence on human immune cells, specifically regarding the activation of dendritic cells (DCs) and the interaction with the autologous cytotoxic T lymphocyte (CTL) clone, was investigated. Melanoma cells were infected with either JX-GFP or TG6002 alone or in combination with 5-FC and 5-FU. The influence of viral infection on cell viability followed a time- and multiplicity of infection dependent manner. Combination of virus treatment with 5-FU resulted in stronger reduction of cell viability. TG6002 in combination with 5-FC did not significantly strengthen the reduction of cell viability in this setting. Expression of calreticulin and high mobility group 1 protein (HMGB1), markers of immunogenic cell death (ICD), could be detected after viral infection. Accordingly, DC maturation was noted after viral oncolysis. DCs presented stronger expression of activation and maturation markers. The autologous CTL clone IVSB expressed the activation marker CD69, but viral treatment failed to enhance cytotoxicity marker. In summary, vaccinia viruses JX-GFP and TG6002 lyse melanoma cells and induce additional immunostimulatory effects to promote antitumor immune response. Further investigation in vivo is needed to consolidate the data. Keywords: oncolytic virus, vaccinia virus, immunogenic cell death, dendritic cells, cytotoxic T lymphocytes, immunotherapy

Cutaneous lymphomas (CLs) are a heterogeneous group of lymphoproliferative disorders that are manageable by immunotherapy. Twenty-one patients were enrolled in a prospective open-label, dose-escalation multicenter study evaluating the effects of repeated TG1042 [adenovirus-interferon (IFN)-gamma] intralesional injections in patients with primary CLs, of which 18 were of T-cell and 3 of B-cell type. Repeated intralesional therapy using TG1042 consistently results in local tumor regressions in about half of treated patients and one-third of patients also in regressions in noninjected distant lesions, likely reflecting the systemic immune activation after intralesional therapy. Treatment was well tolerated with few adverse events including injection site reactions, chills, lymphopenia, and fever. Immune monitoring in the peripheral blood demonstrated systemic immune activation and the induction of antibodies against tumor antigens in some patients without clear association with clinical responses. CLs, in particular B-cell lymphomas with high objective response rates, seem to be excellent targets for this type of immunotherapy.

Purpose: Transfer of prodrug activation systems into tumors by using replication-deficient viruses has been suggested to be an effective method for achieving high local and low systemic anticancer drug concentrations. However, most current suicide gene therapy strategies are still hindered by poor efficiency of in vivo gene transfer, inefficient tumor penetration, limited bystander cell killing effect, and need of large prodrug doses. We hypothesized that local amplification provided by a replication competent platform would help overcome these limitations. Experimental Design: We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Δ24FCU1 expressing the fusion suicide gene FCU1. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Results: We examined the efficacy of Ad5/3-Δ24FCU1 and the replication-defective control Ad5/3-FCU1 with and without 5-FC. FCU1 expression was confirmed by Western blot, whereas enzymatic conversion levels in vitro and in vivo were determined by high-performance liquid chromatography separation. Significant antitumor effect was observed in vitro and in vivo in a murine model of head and neck squamous cell carcinoma. Although we observed a decrease in viral DNA copy number in vitro and in tumors treated with Ad5/3-Δ24FCU1 and 5-FC, suggesting an effect on virus replication, the highest antitumor effect was observed for this combination. Conclusions: It seems feasible and efficacious to combine adenovirus replication to the FCU1 prodrug activation system. Clin Cancer Res; 16(9); 2540–9. ©2010 AACR.

The increasing importance of recombinant adenoviral vectors for gene therapy, cancer therapy, and the development of prophylactic and therapeutic vaccines has led to worldwide efforts toward scalable process development suitable for commercial manufacturing of replication-deficient adenoviral vectors. This review focuses on the manufacturing of adenovirus for clinical trials in the context of good manufacturing practice conditions and regulations.

Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

Liver toxicity and inflammation were assessed in C57BL/6, CBA, and BALB/c mice injected intravenously with a series of recombinant adenoviruses deleted simultaneously in E1/E3, in E1/E3/E2A, or in E1/E3/E4. All vectors were either devoid of transgenes or carried in E1 the human CFTR cDNA under the control of the CMV promoter. Injection of the E1/E3-deleted vector induced a significant liver dystrophy and inflammatory responses that were accompanied by an increased serum transaminase concentration. The vector toxicity remained elevated on additional deletion of the E2A gene and was further enhanced when hCFTR was expressed. In contrast, additional deletion of E4 led to a reduction in hepatotoxicity, suggesting an active role of E4 gene products in liver injury. However, deletion of E4 also led to a loss of transgene expression. To identify the individual E4 product(s) involved in liver toxicity and in the regulation of transgene expression, a series of isogenic E1/E3-deleted vectors, with or without the hCFTR transgene, and containing various combinations of functional E4 open reading frames (ORFs), were evaluated in vitro and in vivo. We demonstrate that liver injury was markedly reduced with vectors containing either ORF3 alone or ORF3,4 while vectors containing ORF4, ORF6,7 or ORF3,6,7 still displayed elevated hepatotoxicity and inflammatory responses. Moreover, transgene expression was restored when ORF3,4 or ORF3,6,7 was retained in the vector. These results highlight the importance of the E4 gene products in the design of improved in vivo gene transfer vectors.

Background Strong and stable transgene expression is fundamental to the success of recombinant adenovirus vectors in human gene therapy. However, control of transgene expression is a complex process, involving both viral and cellular factors. In this study, the influence of the E4 adenoviral region on the activity of various promoters was investigated in vitro and in vivo. Methods Pairs of isogenic E1° and E1°E4° vectors were generated and compared. Levels of transgene expression were determined by Northern blot, ELISA and FACS analysis. Initiation of transcription was studied by nuclear run-on assays. Results Similar to the viral CMV and RSV promoters, the activity of the ubiquitous cellular PGK promoter required the presence of the E4 genes in vitro and in vivo. In contrast, transgene expression from selected liver- and tumor-specific promoters did not require E4 functions. Conclusion Together with the reported low liver toxicity of E1°E4° vectors, the independence of E4 of liver-specific promoters renders such vectors interesting alternatives to the use of gutless vectors. Copyright © 2000 John Wiley & Sons, Ltd.

In a previous study we showed that multiple deletions of the adenoviral regulatory E1/E3/E4 or E1/E3/E2A genes did not influence the in vivo persistence of the viral genome or affect the antiviral host immune response (Lusky et al., J. Virol. 72:2022–2032, 1998). In this study, the influence of the adenoviral E4 region on the strength and persistence of transgene expression was evaluated by using as a model system the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovirus (CMV) promoter. We show that the viral E4 region is indispensable for persistent expression from the CMV promoter in vitro and in vivo, with, however, a tissue-specific modulation of E4 function(s). In the liver, E4 open reading frame 3 (ORF3) was necessary and sufficient to establish and maintain CFTR expression. In addition, the E4 ORF3-dependent activation of transgene expression was enhanced in the presence of either E4 ORF4 or E4 ORF6 and ORF6/7. In the lung, establishment of transgene expression was independent of the E4 gene products but maintenance of stable transgene expression required E4 ORF3 together with either E4 ORF4 or E4 ORF6 and ORF6/7. Nuclear run-on experiments showed that initiation of transcription from the CMV promoter was severely reduced in the absence of E4 functions but could be partially restored in the presence of either ORF3 and ORF4 or ORFs 1 through 4. These results imply a direct involvement of some of the E4-encoded proteins in the transcriptional regulation of heterologous transgenes. We also report that C57BL/6 mice are immunologically weakly responsive to the human CFTR protein. This observation implies that such mice may constitute attractive hosts for the in vivo evaluation of vectors for cystic fibrosis gene therapy.

We have developed and characterized cultures of healthy and dystrophic canine myoblasts for the evaluation of various gene transfer protocols. The number of desmin-positive myoblasts was elevated (>>80%) in cultures of myoblasts obtained from different muscle territories, the diaphragm muscle giving rise to the purest cultures. Myoblasts from dogs turned out to be a very convenient source of well transfectable and transducible cells. Transfection with plasmid DNA allowed efficient transgene expression (50% of β-galactosidase positive cells and about 375 ng luciferase/mg protein after transfection with a calcium phosphate-precipitated plasmid). Infection with high concentrations of adenoviral and retroviral vectors allowed transgene (β-galactosidase or mini-dystrophin) detection in about 75 to 90% of the canine cells. Therefore, primary dog myoblast cultures represent a useful in vitro model for viral and non-viral gene delivery, as well as for functional evaluation and cell grafting with applications in genetic diseases, vaccination or production of circulating therapeutic proteins.

RATIONAL: While a variety of registered therapies exist for Cutaneous T Cell Lymphoma, no such therapy is available for Cutaneous B Cell Therapy. In this context we performed a phase II, open label, multicenter, non-comparative study to evaluate the efficacy and safety of repeated intra-lesional administrations of TG1042 (adenovirus-interferon-γ) in patients with relapsing primary cutaneous B-cell lymphomas (CBCL). METHOD: Thirteen patients have been enrolled and received intralesional injections of TG1042 containing 5×10(10) viral particles into up to six lesions simultaneously. Injections were performed on days 1, 8 and 15 of each of four consecutive 28 day cycles. RESULTS: Eleven (85%) out of 13 enrolled patients showed an objective response after injections of TG1042. Seven patients (54%) exhibited complete and four (31%) displayed partial response. The median time to disease progression in the study population was 23.5 months (range 6.25 to 26+). Most commonly observed adverse events were minor to moderate flu-like symptoms, fatigue and injection site reactions. CONCLUSIONS: Our study showed that treatment with TG1042 was associated with a clinical benefit in the majority of the patients with relapsing CBCL, including tumor regression, a clinically meaningful duration of response and a good treatment tolerance. TRIAL REGISTRATION: www.clinicaltrials.govNCT00394693.

E1, E3-deleted, replication-deficient recombinant adenoviruses are widely studied as vectors for their capacity to transfer therapeutic genes in vivo. They can infect a wide variety of dividing and quiescent cells from different organs and possess a large packaging capacity. One of the major limitations in the use of these vectors for gene therapy is the transient expression of the transgene in vivo and the poor transduction efficiency when re-administered. Despite the deletion of the viral E1 region, low level of early and late viral genes are expressed in vivo. Thus, viral antigens plus those derived from transgene expression in transduced cells contribute to cellular immune responses leading to the destruction of these cells. Production of anti-adenovirus antibodies, the cellular immune response as well as the early non-specific clearance of the vectors, constitute barriers to successful gene therapy. New vectors have been derived with additional deletions in the E2a or the E4 regions. Such second generation vectors were evaluated in vivo. These studies have revealed the complexity of the immune mechanisms elicited by these vectors and the importance of several parameters in these evaluations (i.e. mouse strains, nature of the transgene, route of administration...). In order to inhibit the production of neutralizing antibodies to adenovirus that prevent from further readministration of the vectors, immunosuppressive strategies were undertaken. Treatment regimens with immunosuppressive drugs (cyclophosphamide, FK506) or with monoclonal antibodies that block either the T cell receptor or costimulation pathways allow prolonged transgene expression and/or readministration of adenoviral vectors. In addition, transduction efficiencies may be increased by transiently inhibiting non-specific immune mechanisms that lead to the dramatic early clearance of the vectors. Taken together, these strategies may improve further gene therapy protocols by decreasing the host immune response to adenoviral vectors.

TPS4146Background: Pexa-Vec (pexastimogene devacirepvec; JX-594) is an oncolytic and immunotherapeutic vaccinia virus designed to selectively replicate in and destroy cancer cells. It causes direct oncolysis accompanied by tumor vascular disruption and anti-tumor immunity mediated by expression of the transgene GM-CSF. SOR, a multi-targeted tyrosine kinase inhibitor, is the standard of care for first-line systemic treatment of advanced HCC. Both preliminary preclinical and clinical data suggest complementary anti-tumor effects of a sequential combination of Pexa-Vec followed by SOR possibly by targeting the tumor vasculature via different mechanisms (Heo et al., Mol Ther 2011). A randomized phase II dose-finding study with 3 Pexa-Vec intratumoral (IT) liver injections in first line advanced HCC patients showed an acceptable safety profile and a significant increase in overall survival (OS) in the highest dose group (109pfu) (Heo et al., Nat Med 2013). Methods: This global, randomized, open-label, phase II...

Initiation of bovine papillomavirus (BPV) DNA synthesis in vivo and in vitro depends on the interaction of the viral initiator protein E1 with the replication origin (ori+ DNA). The viral E2 protein assists this interaction, resulting in a cooperative assembly of both proteins on the replication origin. Using gel mobility-shift experiments, we demonstrate that in the presence of both E1 and E2 proteins two classes of ori+ DNA complexes were formed: complex 1 (c1) and complex 2 (c2). Formation of c1 depended on both the E1 and E2 proteins and both proteins were contained within c1. The generation of c2 was dependent on the E1 protein and could be enhanced by E2, but the E2 protein was not detected within c2. At high E2/E1 ratios, c1 was the dominant complex formed. Under these conditions, E1-dependent BPV DNA synthesis in vitro was inhibited. At low E2/E1 ratios, the stimulation of c2 was correlated with the stimulation of BPV DNA replication by E2 in vitro. These data suggest that E2 assists E1 in the formation of an intermediate c1 complex, which is replication inactive. The c1 complex is converted in turn to the replication-active c2 complex, which contains E1 but lacks E2. We propose that the ratios of c1 and c2 formed in response to the levels of E1 and E2 protein determine the potential for BPV DNA synthesis in vitro and in vivo and may contribute to copy number regulation of BPV plasmids within the cell.

Chemical and enzymatic probing techniques were used to examine the interaction of the bovine papillomavirus type 1 E1 and E2 proteins with the viral origin of replication (ori). E1 was found to generate significant distortions to the structure of ori, as assayed by KMnO4 oxidation of DNA. The primary site of ori distortion was located within and adjacent to the AT-element of the core replicator sequence, although a number of minor structural transitions were also detected. The induction of these structural changes required ATP and appeared to require ATP hydrolysis. E2 was found to decrease the amount of E1 required for ori distortion but did not significantly alter the pattern of structural distortion. In contrast, the presence of E2 resulted in a biphasic mechanism for E1 binding to ori, as assayed by nuclease protection. Under these conditions, E1 bound preferentially to the dyad symmetry region containing the conserved Hpa I site. Higher levels of E1 were required for binding to the adjacent ori AT-rich region. Thus, these data suggest that E2 can order the stepwise binding of E1 to ori.

Using quantitative gel retardation assays the properties of the bovine papilloma virus (BPV) origin recognition protein E1 and the effect of the viral E2 protein on the binding of E1 to BPV origin DNA were examined. As reported previously (Seo, Y.S., Mueller, F., Lusky, M., Gibbs, E., Kim, H.-Y., Phillips, B. and J. Hurwitz (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2865-2869), the E1 protein binds specifically to DNA sequences within the BPV origin (ori+) of replication. We also show that the presence of MgCl2 and ATP could stabilize the E1 ori+ DNA complex. At low levels of E1, ori+ DNA binding was greatly stimulated by the viral E2 protein when the intact E2 binding site 12 was present on the DNA. In addition DNA-protein complexes formed in the presence of both E1 and E2 were more stable than those formed with E1 alone. In the absence of an E2 binding site the E2 protein inhibited the binding of E1 to the BPV origin. Spacing of 0 or 9 base pairs between the E1 binding site and the E2 binding site 12 abolished the stimulation of E1-DNA binding by E2, whereas spacing of 6 base pairs between the two binding sites allowed for efficient stimulation. The data presented account for a direct role of E2 in BPV DNA replication. We propose that the cooperative binding of both the E1 and E2 proteins to BPV ori+ DNA is mediated by protein-protein interactions and by protein-DNA interactions, which include the formation of specific contacts of E2 with DNA.

Replication of bovine papilloma virus (BPV) DNA requires two virus-encoded proteins, E1 and E2, while all other proteins are supplied by the host cell. Here, we describe the isolation of the E1 protein and show that it is a multifunctional protein. Purified E1 protein was required for the in vitro replication of BPV origin-containing DNA by extracts of mouse cells, as reported [Yang, L., Li, R., Mohr, I. J., Clark, R. & Botchan, M. R. (1991) Nature (London) 353, 628-632]. In addition, the E1 protein cosedimented with a number of other activities including (i) DNA helicase activity, (ii) BPV origin-containing DNA-specific binding activity, (iii) DNA-dependent ATPase activity, and (iv) BPV origin-specific unwinding of superhelical DNA. The E1 protein, acting as a helicase, moved in the 3'-->5' direction, like simian virus 40 (SV40) large tumor antigen, which plays a pivotal role in SV40 DNA replication. However, unlike the SV40 large tumor antigen, the helicase activity of E1 was stimulated 5-fold by the presence of a fork structure at the junction between single-stranded and double-stranded DNA and was supported efficiently by all eight nucleoside triphosphates. The E1-catalyzed ATPase activity required the presence of single-stranded or double-stranded DNAs.

The 68-kDa bovine papillomavirus (BPV) type 1 replication protein E1 and the 48-kDa transactivator protein E2 form a complex that specifically binds DNA [Mohr, I.J., Clark, R., Sun, S., Androphy, E.J., MacPherson, P. & Botchan, M.R. (1990) Science 250, 1694-1699]. We have confirmed this observation and shown that the E1-E2 complex binds to DNA fragments that contain the BPV plasmid maintenance sequence 1 and a site for the initiation of bidirectional BPV DNA synthesis. The E1 protein was found to bind preferentially to non- or underphosphorylated species of E2, suggesting that the phosphorylation state of E2 modulates the association of the two proteins. Replication-deficient E1 mutants with single amino acid substitutions and deletions in the carboxyl terminus failed to interact with E2, indicating that a region in the E1 carboxyl terminus is required for E1 to interact with E2. Our results suggest that the replication deficiency of some E1 mutants reflects their inability to associate with E2.

The bovine papillomavirus genome contains two cis-acting sequences which can serve as signals for replication. At least three virally encoded genes seem to be involved in plasmid replication: E6, E6/7 and E1. Mutations in either the E6 or the E7 open reading frame create plasmids that are maintained at a low copy number per cell. Mutations in the E1 open reading frame are absolutely lethal to replication. Complementation experiments show that these mutations define separate genes. Experiments are described which show that cells harbouring plasmids with mutations in either the E6 or the E7 open reading frame acquire an immunity to high copy-number plasmids. We suggest that either the cell or the virus encodes a repressor. The positive action of E6 and E6/7 modulates the activity of this repressor to allow for the high copy-number state. Though the viral oncogenes are capable of transforming cells separately when they are expressed as part of certain recombinant DNA expression systems, it is clear that, in the context of the entire viral replicon, interactions between the transforming functions and replication functions must exist.

(IF : 4,608); International audience; Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. The aim of our study was to develop an Ad vector expressing a biologically active green fluorescent protein (GFP)- CFTR chimera that can be tracked by both its localization and chloride channel function. No study thus far has demonstrated a GFP-CFTR construct that displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CFKM4 (CFTRDF508), as well as human airway epithelial cells from a patient with cystic fibrosis (CF-HAE) and from a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/ GFP-CFTRwt and Ad5/GFP-CFTRDF508. The GFP-CFTRwt protein expressed was efficiently addressed to the plasma membrane of tracheal cells and to the apical surface of polarized CF-HAE cells, while GFP-CFTRDF508 mutant was sequestered intracellularly. The functionality of the GFP-CFTRwt protein was demonstrated by its capacity to correct the chloride channel activity both in CF-KM4 and CF-HAE cells after Ad transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55 to 70% transduction resulted in 70% correction of the Cl2 channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl2 transport. We show for the first time a GFP-CFTR chimera that localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as nontagged CFTR.

Previously, we have identified a tumor cell-specific peptide, HEW, by panning of phage display libraries on the human colorectal cancer cell line WiDr. In this report we demonstrate that this peptide can modify the infection properties of adenovirus vectors. Increased infectivity of replication-deficient adenovirus 5 vectors in WiDr cells was observed upon genetic insertion of the HEW peptide in the HI loop of the fiber knob. Moreover, whereas the coxsackie and adenovirus receptor (CAR)-ablating fiber mutation S408E abolished apparent infection in CAR-positive WiDr cells, the insertion of HEW completely restored infectivity toward these cells in vitro. To assess whether the de- and re-targeted infection profile was maintained in vivo, the fiber-modified adenovirus vectors were injected intratumorally or intravenously in WiDr tumor-bearing Swiss nu/nu mice. No significant differences in efficiency of infection could be observed suggesting alternative viral uptake mechanisms in vivo. Next, we have included the fiber shaft mutation S(*) in our studies, which was described to confer a de-targeted phenotype in vivo. Reduced gene transfer due to the S(*) mutation both in vitro and in vivo could be confirmed. Insertion of HEW in the HI knob loop of shaft-mutated fiber, however, did not rescue infectivity in target cells neither in vitro nor in vivo. We demonstrate the efficient ligand-mediated re-targeting of adenoviral vector infection to the human cancer cell line WiDr. The lack of apparent re-targeting in the in vivo situation is described.

TPS4161^ Background: Pexa-Vec is a targeted oncolytic and immunotherapeutic vaccinia virus engineered to express human granulocyte-macrophage colony stimulating factor (GM-CSF). Direct oncolysis plus GM-CSF expression stimulates tumor vascular disruption and anti-tumor immunity (Nature Rev Cancer, 2009). Pexa-Vec was well-tolerated in Phase 1 trials and was shown to replicate in metastatic tumors following intratumoral (IT) or intravenous (IV) administration (Lancet Oncol, 2008 and Nature, 2011). A randomized high vs low dose Phase 2 trial in 30 patients with advanced HCC, demonstrated prolonged survival in the high-dose Pexa-Vec arm (median survival 14.1 mo vs. 6.7 mo; Hazard Ratio 0.39, p=0.02) (AASLD Annual Meeting, 2011, LB1). Methods: TRAVERSE is a Phase 2b randomized, open-label, multi-center trial in patients with advanced HCC who have failed sorafenib treatment. Approximately 120 patients will be randomized 2:1 to Pexa-Vec plus BSC versus BSC, respectively. Randomization will be stratified by region (Asian vs. non-Asian); sorafenib intolerant vs refractory; and presence vs absence of extra-hepatic disease. The primary objective is to determine overall survival. Main inclusion criteria are advanced HCC having failed sorafenib (intolerance or radiographic progression during or < 3 months following last sorafenib), Child-Pugh A-B7 (no ascites), acceptable hematologic function. Assuming a median overall survival of 4.0 months with BSC and a target hazard ratio of 0.57 (corresponding to an experimental arm median survival of 7.0 months), 73 events (deaths) will provide 70% power at 1-sided alpha = 0.05 to detect a difference in overall survival between the treatment groups using a stratified logrank test. Patients randomized to Pexa-Vec will receive a dose of 109 plaque forming units (pfu) IV on Day 1 followed by five IT treatments between Day 8 and Week 18. Enrollment has begun on this study with clinical trial registry number of NCT01387555. Clinical trial information: NCT01387555.

TPS4152 Background: JX-594 is a first-in-class targeted oncolytic poxvirus designed to selectively replicate in and destroy cancer cells with epidermal growth factor receptor (EGFR)/ ras pathway activation. Direct oncolysis plus GM-CSF expression stimulates tumor vascular disruption and anti-tumor immunity (Nature Rev Cancer 2009). JX-594 was well-tolerated in Phase 1 trials and was shown to replicate in metastatic tumors following intratumoral (IT) or intravenous (IV) administration (Lancet Oncol 2008 and Nature 2011). A randomized dose-finding Phase 2 trial has been completed with JX-594 in 30 patients with advanced HCC. Treatment with high-dose JX-594 was associated with prolonged survival vs low-dose JX-594 (median survival 14.1 mo vs 6.7 mo; Hazard Ratio 0.39, p=0.02) (AASLD Annual Meeting, 2011, LB1). Methods: TRAVERSE is a Phase 2b randomized, open-label, multi-center trial of JX-594 plus BSC versus BSC in patients with advanced HCC who have failed sorafenib treatment. Approximately 120 patients will be randomized 2:1 to experimental and control arm respectively. Randomization will be stratified by region (Asian vs non-Asian); sorafenib intolerant vs refractory; and presence vs absence of extra-hepatic disease. The primary objective is to determine overall survival in the 2 arms. Assuming a control median overall survival of 4.0 months and a target hazard ratio of 0.57 (corresponding to an experimental arm median survival of 7.0 months), 73 events (deaths) will provide 70% power at 1-sided alpha = 0.05 to detect a difference in overall survival between the treatment groups using a stratified logrank test. Patients randomized to JX-594 will receive a dose of 109 plaque forming units (pfu) IV on Day 1 followed by five IT treatments between Day 8 and Week 18. Main inclusion criteria are advanced HCC having failed sorafenib (intolerance or radiographic progression during or < 3 months following last sorafenib), Child-Pugh A-B7 (no ascites), acceptable hematologic function. Enrollment has begun on this study with clinical trial registry number of NCT01387555.

The replication of bovine papilloma virus (BPV) DNA in vivo requires two viral-encoded proteins, E1 and E2, while all other proteins are derived from the host. We described previously the isolation of the E1 protein and showed that it contains multiple functions required for BPV DNA replication. The BPV transcription factor E2 was shown by others to stimulate BPV DNA replication in vitro. Here, we present results that account for the role of the E2 protein. The E1 protein bound selectively to the BPV minimal origin of replication. This process required MgCl2 and ATP for maximal efficiency. The E1 protein also catalyzed a BPV origin-dependent DNA unwinding reaction. In this report, we show that at low levels of E1 protein, origin binding could be stimulated up to 40-fold by the E2 protein, provided that the DNA contained an E2 binding site. Consistent with this result, the E2 protein stimulated the origin-specific unwinding reaction catalyzed by E1, but it had no effect on the nonspecific E1-catalyzed helicase activity. In the absence of an E2 binding site, both origin-dependent binding and unwinding reactions with the E1 protein were unaffected by the E2 protein. These results suggest that E2 participates in the initiation of BPV DNA replication by enhancing E1 binding to the BPV origin via DNA-protein and protein-protein interactions.

Bovine Papilloma Virus (BPV-1) establishes itself as a multicopy nuclear plasmid in somatic mammalian cells in culture. We report here that two discontinuous regions within the viral genome can independently support extrachromosomal replication of the Tn5 neomycinr gene in cells that provide viral factors in trans. The viral plasmid maintenance sequences (PMS) act in cis and will integrate along with the marker gene in cell lines that do not provide BPV-1 gene products. PMS-1 is localized within a 521 bp region upstream of the BPV-1 early transcription unit; PMS-2 has been localized to a 140 bp region within the putative reading frame for the E1 protein of the viral genome. Recombinant plasmids carrying either of the PMS elements are unrearranged and stably maintained at a constant copy number supernumerary to the resident BPV-1 genomes even in the absence of selective pressure. Specific deletion mutants within the viral genome show that BPV-1 gene products required for morphological transformation are dispensable for plasmid maintenance. In mouse cells cotransformed with such deletion derivatives and an unlinked marker gene (neomycinr or Tk), the marker genes integrate into the host genome while the BPV molecules are nonselectively carried as nuclear plasmids. This result implies that the BPV-1 genome must have signals that specifically preclude integration in the presence of transacting factors.

In pBR313-lambda dv hydrid plasmids a second system for initiation of DNA replication has been detected in lambdoid replicator DNAs (in the absence of the p0 promoter). The "maximal" (or "maxi") initiation system depends on the origin of replication (ori) sequence, in conjuction with the "inceptor" (ice) element located in the lambdoid cII genes. Only leftward, but not bidirectional, primer RNA synthesis seems to be initiated at ori in its newly defined boundaries, and it appears to be catalysed by dnaG-coded primase. Only if transcriptionally activated, will ori effectively initiate lambda specific, O and P-dependent "maximal" hybrid-plasmid replication. In addition, it will repress a complete lambda "minimal" initiation system in cis, i.e., if present on the same plasmid molecule. This newly discovered repressive activity of the ori system depends on only three factors: an intact left section of ori, the O product, and transcriptional activation of ori (rightward or leftward). A repressed minimal initiation system will regain its activity as soon as a segment carrying either part of the O gene or a promoter for transcriptional activation is delected from such a plasmid which was combining both the "mini" and "maxi" systems of lambda replication.

SV40 DNA replication requires interactions between proteins provided by the host cell, the virus-coded A protein and a viral sequence of ∼80 base pairs (bp), which serves as an origin of DNA replication1,2. The apparent simplicity of this virus has provided a useful means of producing3,4 SV40 virions that carry specific eukaryotic genes which can infect cells in culture and replicate to a high copy number. One disadvantage to this approach is that the viral DNA sequences necessary for replication and the gene of interest must be precisely tailored to package efficiently into SV40 virus particles. In an attempt to circumvent this limitation, we have studied the replication of SV40–pBR322 plasmids after transfection into simian cells with the ultimate goal of using the viral replicon as a means of amplifying the expression of cloned fragments inserted in these vectors. Such a dual-host replicon also offers the possibility of rescuing and subsequently cloning genes which are properly expressed and stably integrated into the cell genome5. We and others (refs 5–8 and Schaffner, personal communication), have noted that such plasmids propagated in Escherichia coli replicate poorly if at all after transfection of simian cells. Further-more, recombinant plasmids isolated from the transfected simian cells subsequently show a reduced ability to retransform E. coli5. We now demonstrate that both these phenomena are related to the presence of a cis-acting DNA sequence in the bacterial vector, pBR322. SV40–pBR322 plasmids lacking this sequence replicated as effectively as authentic SV40 DNA in simian cells and were re-established as plasmids in E. coli as effectively as plasmid DNAs extracted from E. coli.

A comparison of the available lambdoid origin sequences allows to point out several structural features in common with the DNA sequence of the phage G4 intercistronic region between genes F and G, which can be interpreted as recognition elements of a primase promoter sequence. These leading strand initiation signals are located in leftward orientation on the A-rich strand of all lambdoid origins, and by comparison with G4 require a single-stranded binding sequence, which appears to result from transcriptional activation. Binding of 0 protein to the origin interferes with transcription across ori , which therefore, has to precede these ori/0 interactions.

Hybrid plasmid dissection and reconstruction pro-cedures have shown that two different signal structures for initiation of DNA replication can be separated within the replicator region of various lambdoid phages. From our hybrid plasmid experiments we conclude that the origin of replication (ori) serves as the initiation site for leftward primer RNA synthesis while the replicational inceptor (iae) signal controls termination of that primer RNA and inception of daughter strand DNA synthesis. Hybrid plasmids containing only ori are unable to initiate lambdoid replication, whereas plasmids containing iae in connection with a leftward promoter do show replicational activity (minimal replication system).

A transient assay has been used to study bovine papilloma virus type 1 (BPV-1) replication. We show that BPV-1 early replication occurs faster than cellular DNA synthesis. Initial replication events are dependent on a gene product(s), encoded by the BPV-1 E1 open reading frame. Mutational analysis of the viral upstream regulatory region shows the requirement of two domains in cis for replication. Domain one, located outside of the viral 69% transforming fragment, is an enhancer-like activity and can be replaced by other known viral enhancers. Domain two lies within sequences previously defined as plasmid maintenance sequence 1. The apparent requirement for a proximal enhancer function for replication may explain why certain BPV-1 constructions, when linked to bacterial plasmid sequences, can be maintained extrachromosomally while others cannot.

Cells transformed with bovine papilloma virus type 1 mutants in the E6 or E6/7 genes are resistant to high-copy-number amplification of wild-type DNA after supertransfection. Transient and stable replication assays demonstrate this effect. If the supertransfected DNA has a mutation in a newly defined gene (M), this cellular immunity to high-copy-number replication is overcome, resulting in transient replication of the input DNA. In contrast, the resident plasmid does not participate in amplification and is maintained at a constant low copy number. Cotransformation of M- mutants and wild-type DNA into these cells leads to shutoff of replication of both genomes. Thus, M- mutants define a trans-acting negative modulator that regulates viral replication. This function is distinct from the positive factors required for replication. We propose a model that explains why the loss of E6 and E6/7 function leads to immunity of the infected cell.