No abstract

Development of immunocompetent patient-like models that allow direct analysis of human adenovirus-based conditionally replicative adenoviruses (CRAds) would be beneficial for the advancement of these oncolytic agents. To this end, we explored the possibility of cross-species replication of human adenovirus type 5 (Ad5) in canine cells. With a panel of canine tumor cell lines of both epithelial and mesenchymal derivations, we demonstrate that human Ad5 can productively infect canine cells. Since the biological behavior and clinical presentation of certain dog tumors closely resemble those of their human counterparts, our results raise the possibility of exploiting canine models for preclinical analysis of candidate CRAd agents designed for human virotherapy.Conditionally replicative adenovirus (CRAd) agents represent a promising new therapeutic approach for cancer. This strategy is based on the application of an adenovirus engineered to selectively replicate in tumor targets (1, 3). This tumor-selective replication forms the functional basis of the antineoplastic effect achieved by direct target cell killing in a process termed oncolysis (1, 16). The exceptional promise of these agents has predicated their rapid transition to human phase I clinical trials whereby the overall safety of this approach has been validated (15,22,28). Nonetheless, the very limited indications of efficacy to this point have established the requirement for further design advances to enhance CRAd antitumor potency (11,15,20,23).Critical to the derivation of advanced-generation CRAds is the development of model systems capable of delineating key therapeutic indices. For CRAds, the fact that human adenoviruses can accomplish only abortive replication in murine targets (7-9, 17) has restricted the preclinical toxicity information that may be derived from SCID-xenograft model systems typically used for efficacy analysis. Further, these immunologically incompetent models cannot provide useful information with respect to CRAd immunobiology and vector-host interactions. On this basis, it is clear that there exists a field-wide need for immunocompetent syngeneic models for full analysis of candidate CRAd agents. Several recent reports have described model systems to achieve these goals. Hemminki et al. have exploited the availability of human-like canine models of cancer (31) for the evaluation of CRAd agents derived from canine adenoviruses (12,15,31). This approach potentially allows the study of vector-host interactions in an immunocompetent host in the context of a patient-like cancer model. CRAd constructs based on canine adenoviruses, however, may embody substantial biological differences from human adenovirus-based CRAds. Alternatively, an approach has been proposed whereby adapted murine cells permissive for limited human adenovirus replication are transplanted into an immunocompetent mouse host (10,29). Whereas this approach may allow direct analysis of human adenovirus-based CRAds in a murine system, this transplant model does not repre...

The full realization of conditionally replicative adenoviruses (CRAds) for cancer therapy has been hampered by the limited knowledge of CRAd function in vivo and particularly in an immunocompetent host. To address this issue, we previously proposed a canine adenovirus type 2 (CAV2)-based CRAd for clinical evaluation in canine patients with osteosarcoma (OS). In this study, we evaluated infectivityenhancement strategies to establish the foundation for designing a potent CAV2 CRAd with effective transduction capacity in dog osteosarcoma cells. The results indicate that the native CAV2 fiber-knob can mediate increased binding, and consequently gene transfer, in both canine osteosarcoma immortalized and primary cell lines relative to previously reported Ad5 infectivity-enhancement strategies. Gene delivery was further enhanced by incorporating a polylysine polypeptide onto the carboxy terminus of the CAV2 knob. This vector demonstrated improved gene delivery in osteosarcoma xenograft tumors. These data provide the rationale for generation of infectivity-enhanced syngeneic CAV2 CRAds for clinical evaluation in a dog osteosarcoma model.

Conditionally replicative adenoviruses (CRAds) are engineered to replicate only in the target tissue and destroy tumor through their cytopathic effect. Because of restricted in vivo replication, it is difficult to model behavior of human Ad5-based vectors in animal subjects. To circumvent this, we developed a "syngeneic" canine CRAd based on canine adenovirus type 2 (CAV2) transcriptionally targeted to canine osteosarcoma (OS) cells. Canine OS is an outstanding model of human OS and is the most common primary bone tumor of dogs. Because conventional therapies extend median survival by approximately 6-8 months, canine OS remains a serious therapeutic challenge shared by human OS patients. Prior to using any CRAd for clinical trials in dogs, we sought to examine the effects and safety of administration of OS-targeted CAV2 CRAd in normal dogs. Short-term physiologic indicators of stress and shock, as well as gross and histological changes in a variety of tissues, were examined, and no major signs of virus-associated toxicity were noted. In addition, short-term immunosuppression did not increase CRAd toxicity. This study marks the first administration of a CRAd in an outbred large animal model and is an important milestone in the application of this modality in human patients.

Adenovirus serotype 5 (Ad5) vectors containing Ad B-group fibers have become increasingly popular as gene transfer vectors because they efficiently transduce human cell types that are relatively refractory to Ad5 infection. So far, most B-group fiber-containing vectors have been first-generation vectors, deleted of E1 and/or E3 genes. Transduction with these vectors, however, results in viral gene expression and is associated with cytotoxicity and immune responses against transduced cells. To circumvent these problems, we developed fiber-chimeric Ad vectors devoid of all viral genes that were produced either by the homologous recombination of first-generation vectors or by using the Cre/lox-based helper virus system. In this study we compared early steps of infection between first-generation (35-kb genome) and Ad vectors devoid of all viral genes with genome sizes of 28 kb and 12.6 kb. All vectors possessed an Ad35-derived fiber knob domain, which uses CD46 as a primary attachment receptor. Using immortalized human hematopoietic cell lines and primary human CD34-positive hematopoietic cells, we found that the Ad genome size did not affect the efficiency of virus attachment to and internalization into cells. Furthermore, independently of the genome length and structure, all vectors migrated to the nucleus through late endosomal and lysosomal cellular compartments. However, the vector containing the short 12.6-kb genome was unable to efficiently escape from endosomes and deliver its DNA into the nucleus. Moreover, compared to other vectors, these Ad particles were less stable and had an abnormal capsid protein composition, including a lack of capsid-stabilizing protein IX. Our data indicate that the size and structure of the packaged viral genomes can affect the integrity of Ad particles, which in turn results in lower infectivity of Ad vectors.