2990 Pancreatic cancer is most often diagnosed at an unresectable stage due to metastasis. Thus, there is a high demand for the development of novel therapeutic modalities for systemic usage. While conditionally replicative adenovirus (CRAd) is promising candidate, poor expression of adenoviral primary receptor has been a large obstacle for getting requisite tumor transduction in this tumor context. We have reported that the incorporation of RGD4C motif into adenoviral HI-loop (RGD modification) and substitution of Ad5 fiber-knob region with Ad3 knob (Ad5/3 modification) enhances the CRAd efficiency upon intratumoral injection. However, it has been questioned if the infectivity enhancement is really beneficial for in vivo anti-tumor effect in the condition closer to the patient situation. In this study, we analyzed the effect of the adenoviral fiber modifications upon systemic vector administration by using orthotopic pancreatic cancer xenograft model. In order to analyze tumor transduction efficiency, the luciferase expression vectors with normal-Ad5, RGD-modified, and Ad5/3 modified fibers were employed. The cyclooxygenase (COX)-2 promoter-based CRAd and its fiber modified versions were used for the analyses of in vivo anti-tumor effect. To establish the orthotopic pancreatic cancer model, an Hs766T subcutaneous xenograft was established in a nude mouse first. Then, the small pieces (1mm 3 ) made from this parental tumor were transplanted into pancreatic bed of other nude mice. The viruses were injected from tail vein 3 days after tumor transplantation. For transduction assay, luciferase activity was analyzed 2 days after vector administration. For the anti-tumor effect, the mice were sacrificed 28 days after CRAd administration. In vitro , 5/3 modified CRAds exhibited remarkably higher viral replication compared to fiber unmodified counterparts in pancreatic cancer cell lines. As for in vivo tumor transduction, only Ad5/3 modified vector showed significantly (8 times) higher transduction compared to the background. This tumor transduction enhancement was also observed in pancreatic cancer subcutaneous xenografts upon systemic vector administration. When CRAds were injected systemically to assess the therapeutic effect in the mice with orthotopic tumors, the volume of the tumor treated with 5/3 CRAd COX-2 was significantly lower than that with non-replicative control virus, and comparable to non-selectively replicating wild type Ad. In the harvested tumors, the viral copy number of the 5/3 COX-2 CRAd was significantly high, even in comparison with non-selective wild type Ad. In conclusion, Ad5/3 modification enhanced the therapeutic effect of systemically administered CRAd. This result suggests the benefits of adenoviral tropism modification as a way to achieve therapeutic effect in clinically relevant settings.
The adenovirus (Ad) is a useful vector for cancer gene therapy due to its unparalleled gene transfer efficiency to dividing and quiescent cells. Primary cancer cells, however, often have highly variable or low levels of the requisite coxsackie-adenovirus receptor (CAR). Also, assessment of gene transfer and vector persistence has been logistically difficult in human clinical trials. We describe here two novel bicistronic adenoviral (Ad) vectors, AdTKSSTR and RGDTKSSTR, which contain the herpes simplex virus thymidine kinase gene (TK) for molecular chemotherapy and bystander effect. In addition, the viruses contain the human somatostatin receptor subtype-2 gene (SSTR2), the expression of which can be noninvasively imaged. We enhanced the infectivity of RGDTKSSTR by genetically incorporating the RGD-4C motif into the HI-loop of the fiber. This allows the virus to circumvent CAR deficiency by binding to αvβ3 and αvβ5 integrins, which are highly expressed on most ovarian cancers. The expanded tropism of RGDTKSSTR results in increased infectivity of purified primary ovarian cancer cells and allows enhanced gene transfer in the presence of malignant ascites containing anti-Ad antibodies. RGDTKSSTR may be a useful agent for treating ovarian cancer in clinical trials.
Adenoviral vectors have demonstrated great potential for gene therapy of many diseases. Significant effort has been devoted in this field to achieve targeted gene delivery to specific cells with the intention of improving the therapeutic index and safety. These targeting strategies require a powerful vector detection system to assess vector function. Present methods used to localize vectors are inadequate for evaluating targeting activity. Post-transduction reporter gene expression cannot accurately represent the physical biodistribution of vectors; vector labeling with chemical substrates may affect the infectivity of virions; and probing for viral components requires extensive preparation and strong accessible signal. The field of virotherapy has further challenged the effectiveness of current vector detection systems. Due to the dynamic nature of replicative agents, current static detection techniques cannot be used to monitor replication and spread of these viruses, two key functions of the ideal replicative agent. With respect to the former issue of vector detection in targeting schemes, we sought to develop a vector labeling approach to directly detect adenoviral particles. We designed a genetic labeling system that would also fulfill the latter goal of dynamic monitoring of adenoviral replication and spread. We hypothesized that an adenoviral structural protein genetically fused to an imaging ligand would allow vector detection and serve as a signature of viral replication and localization. Recently we successfully generated and characterized a recombinant adenovirus labeled with capsid fusion pIX-EGFP. The fluorescent label minimally perturbed viral function and provided an optical property useful for vector detection in tracking of cellular infection, flow cytometry, and tissue sections following systemic administration. We expanded our labeling strategy further to produce bioluminescent adenoviruses. The rationale for this pursuit revolves around the fact that bioluminescence can be detected much deeper in tissues than fluorescent proteins with much greater sensitivity and dynamic range as well as virtually no background luminescence. We generated bioluminescent adenoviruses by incorporating Renilla luciferase (RL) and firefly luciferase (FL) onto the pIX locale. Although these two proteins were significantly larger than EGFP, both Ad-IX-RL and Ad-IX-FL were rescued. After propagation and CsCl centrifugation of these two viruses, the viral gradients were fractionated and analyzed for viral DNA content and luciferase activity. Bioluminescence peaks corresponding with the viral DNA were detected for the mature (bottom) and empty capsid (top) viral bands of Ad-IX-RL and Ad-IX-FL, indicating the retained activity of both luciferases not only in the context of a fusion with pIX but also physical association with the virus capsid. We are characterizing these vectors further and are evaluating their general utility as well as potential for noninvasive detection in mice. Bioluminescent adenoviruses generated with our genetic labeling system will have great implications in vector targeting studies and can potentially be used to monitor adenovirus replication and spread in virotherapy applications.
ABSTRACT The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.
Supplementary Material from Mitogen-activated protein kinase kinase 1/2 inhibitors and 17-allylamino-17-demethoxygeldanamycin synergize to kill human gastrointestinal tumor cells <i>in vitro</i> via suppression of c-FLIP-s levels and activation of CD95
2991 Conditionally replicative adenovirus (CRAd) is useful as a cervical cancer gene therapy strategy which seeks to achieve selective viral replication in tumor cells. Oncolysis of infected cells creates a “bystander effect”, leading to infection of neighboring cancer cells with progeny virus. However, one of the obstacles in CRAd clinical trials is the lack of a non-invasive monitoring procedure of CRAd replication and/or spread, thus hampering the understanding of CRAd biology in both an in vitro and in vivo setting. Current detection strategies, including reporter gene expression and viral component detection have been applied to analyze adenoviral vectors; however, these methods are inadequate for assessing transductional targeting. The objective of this proposal was to establish an efficient method for monitoring CRAd replication/spread while retaining CRAd-based therapeutic efficacy for cervical cancer. We have developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and enhanced red fluorescence protein (RFP). The human telomerase reverse transcriptase (hTERT) promoter, which is active in many cancers but mostly inactive in normal organs including the liver, maintains this useful profile in our CRAd construct for cervical cancer. We employed an hTERT promoter-based CRAd with a pIX-RFP mutation (AdhTERT E1-IX-RFP) to evaluate the hTERT-selective oncolysis in in vitro , which conventional hTERT-CRAd with wild type pIX can demonstrate. Then, we examined the correlation between red fluorescence and DNA replication and oncolysis, which are determined by viral E4 DNA copy number and MTS assay, respectively, in both hTERT-negative fibroblasts and WI-38 cells and hTERT-positive cervical cancer cell lines. RFP-labeled hTERT-CRAd showed effective oncolysis in cervical cancer cell lines but not in WI-38. Red fluorescence imaging paralleled both DNA replication and oncolysis in cervical cancer cells, whereas no fluorescence, DNA replication nor oncolysis was detected in WI-38 cells. Moreover, oncolysis and red fluorescence signal were observed in cervical cancer xenograft models. These results suggest that RFP-labeled hTERT-CRAd demonstrated selective and effective oncolysis, which can be monitored fluorescently. This fluorescently labeled hTERT-CRAd may thus contribute to the gene therapy of cervical cancer.
