Search

Replication deficient human adenovirus type 5 (hAd5) is an important gene delivery vehicle and has been used in various fields of biomedical sciences such as gene therapy, cancer therapy and vaccination. Inspite of its various useful features, emergence of replication competent adenovirus (RCA) in recombinant virus stocks is a great concern. In the present study, recombinant adenovirus expressing foot-and-mouth disease virus serotype-O capsid proteins was propagated in HEK-293 cells and purified by CsCl density gradient ultracentrifugation. The virus was serially passaged in HEK-293 cells and at passage level (P) 2–4, 6, 8 and 12, tested for the presence of RCA. A vector dose of 2 × 10 8 and 1 × 10 10 TCID 50 of the virus was used in cell line bioassay and PCR, respectively. Our results demonstrated that the PCR is more sensitive and rapid technique for the detection of RCA in recombinant adenovirus stocks as early as at P3, whereas the bioassay detected the RCA at P8. [ABSTRACT FROM AUTHOR]

Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, and can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1×108, 1×109, or 1×1010 viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1×109 vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. [ABSTRACT FROM AUTHOR]

Abstract: Adenoviral vectors are highly efficient at transferring genes into cells and are broadly used in cancer gene therapy. However, many therapeutic genes are toxic to vector host cells and thus inhibit vector production. The truncated form of E2F-1 (E2Ftr), which lacks the transactivation domain, can significantly induce cancer cell apoptosis, but is also toxic to HEK-293 cells and inhibits adenovirus replication. To overcome this, we have developed binary- and single-vector systems with a modified tetracycline-off inducible promoter to control E2Ftr expression. We compared several vectors and found that the structure of expression cassettes in vectors significantly affects E2Ftr expression. One construct expresses high levels of inducible E2Ftr and efficiently causes apoptotic cancer cell death by activation of caspase-3. The approach developed in this study may be applied in other viral vectors for encoding therapeutic genes that are toxic to their host cells and/or inhibit vector propagation. [Copyright &y& Elsevier]

Abstract: Objective.: Previous studies have demonstrated the pivotal roles of matrix metalloproteinases (MMPs) in cervical cancer progression. Tissue inhibitor of metalloproteinases-3 (TIMP-3) can inhibit all of MMPs, and its overexpression can retard many kinds of tumor growth. Especially, a potent bystander effect would make it to be more advisable for treating cervical cancer whose primary growth pattern is to invade adjacent structures. These characters prompted the authors to explore its further applicability in human cervical cancer. Methods.: We constructed a replication deficient adenoviral vector carrying TIMP-3 (Ad-TIMP-3). It was transferred into different cervical cancer cell lines in vitro and was injected into the tumor xenografts of nude mice in vivo. Results.: Overexpression of TIMP-3 by adenovirus vector arrested cervical cancer cells in G2/M phase. It also promoted growth inhibition of cancer cells by bystander effects. Ad-TIMP-3 infection produced a more potent cell killing effect than Ad-p53 (P <0.05). A synergic effect was observed when Ad-TIMP-3 and cisplatin (cDDP) were used in combination (D <1). In vivo, Ad-TIMP-3 could inhibit cervical cancer xenografts growth. Compared with Ad-TIMP-3 and cDDP groups, tumor growth in Ad-TIMP-3 combined with cDDP group was inhibited more significantly (P <0.05). Conclusion.: These findings indicated that Ad-TIMP-3 bear a therapeutic potential for cervical cancer. [Copyright &y& Elsevier]

Abstract: Virotherapy of cancer using oncolytic adenoviruses has shown promise in both preclinical and clinical settings. One important challenge to reach the full therapeutic potential of oncolytic adenoviruses is accomplishing efficient infection of cancer cells and avoiding uptake by normal tissue through tropism modification. Towards this goal, we constructed and characterized an oncolytic adenovirus, carrying mutated capsid proteins to abolish the promiscuous adenovirus native tropism and encoding a bispecific adapter molecule to target the virus to the epidermal growth factor receptor (EGFR). The new virus displayed a highly selective targeting profile, with reduced infection of EGFR-negative cells and efficient killing of EGFR-positive cancer cells including primary EGFR-positive osteosarcoma cells that are refractory to infection by conventional adenoviruses. Our method to modify adenovirus tropism might thus be useful to design new oncolytic adenoviruses for more effective treatment of cancer. [Copyright &y& Elsevier]

Abstract: Objectives. : Current virotherapy strategies for ovarian cancer have been hampered by limitations in target cell infectivity and nonspecific tissue replication. In an effort to circumvent these limitations, we evaluated various CRAds modified to incorporate novel capsid targeting motifs (RGD and chimeric Ad5/3) with a novel tissue-specific promoter (CXCR4). Methods. : Two novel CRAds (Ad5-CXCR4-F5/3 and Ad5-CXCR4-RGD) were constructed via homologous recombination and verified by PCR and DNA sequencing. The infectivity and viral replication rates of these two CRAds were analyzed via quantitative real-time PCR (QRT-PCR) in cell line experiments using three ovarian cancer cell lines (SKOV3.ip1, Hey, and OV4) and compared to that achieved with a clinical grade CRAd (Δ24-RGD) to be evaluated in a Phase I trial. Cytocidal effects were determined by crystal violet staining in these same cell lines infected with different concentrations of viral particles per cell (0, 0.1, 1, 10, 100, and 500). Additionally, viral replication was evaluated by QRT-PCR in primary ovarian cancer tissue slices from multiple patients with ovarian cancer as well as in primary human normal liver tissue slices in order to establish CRAd selectivity. All experiments incorporated appropriate controls and repeated in triplicate. Results. : Compared to RGD-capsid CRAds (Δ24-RGD and CXCR4-RGD), the F5/3-capsid CRAd (CXCR4-F5/3) demonstrated significant improvements in infection rates (p =0.025, 0.006, and 0.006) in all ovarian cancer cell lines tested (SKOV3.ip1, Hey, and OV4, respectively). In addition to improved transduction of virus into the cells, the TSP CXCR4-based CRAds demonstrated improved viral replication. Specifically, CXCR4-F5/3 further enhanced viral replication 89-fold (p =0.009, 0.010, 0.003) in the same cancer cell lines. Furthermore, CXCR4-F5/3 showed a 4-log improvement in oncolytic potential over Δ24-RGD. In the ex vivo primary ovarian tissue slices, CXCR4-F5/3 showed a 58-fold improvement in viral replication (p =0.005) compared to the clinical grade Δ24-RGD. Both CXCR4-F5/3 and CXCR4-RGD demonstrated significant reduction of viral replication in normal liver slices (p =0.001). Conclusions. : These data suggest that a dual targeted approach is feasible for the combined enhancement of infectivity and replication in ovarian cancer with a specificity that was attenuated in normal liver tissues. In fact, CXCR4-F5/3 outperformed our best CRAd agent to date nearly 60-fold in our most stringent ex vivo model of primary ovarian cancer tissue slices and suggests that this novel agent could be useful for the treatment of ovarian cancer. [Copyright &y& Elsevier]