Multilevel Targeting of Canine Adenovirus Type 2 (CAV2) to Expand Oncolytic Virotherapy in Canine Tumors
Type of DegreePhD Dissertation
General Veterinary Medicine
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Oncolytic virotherapy is a promising therapeutic approach designed to selectively eradicate cancer cells but to spare normal cells. Oncolytic adenoviruses (Ad) are an outstanding and common vehicle for efficient cancer gene therapy. Selection of appropriate intermediate animal models is a basic requirement for successful cancer virotherapy. Some unique characteristics such as inter-individual and intratumoral heterogeneity, similar genomic sequence and instability to humans, and similar environmental exposures validate the dog as a suitable intermediate animal model of cancer and other complex human diseases. Although adenoviruses are the most commonly used viral vector for gene therapy, several concerns, such as normal cell/organ toxicity, lack of suitable cell surface receptors to allow viral entry to the desired cancer cell, and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of these vectors. Hence, new approaches to improve the transductional and transcriptional targeting efficacy of Ad are required for adapting the infectivity of adenovirus in disseminated canine tumors such as multicentric lymphoma, which can serve as a model for human non-Hodgkin’s lymphoma (NHL). Our goal is to explore mechanisms to target canine tumors at both the level of transduction (and infection) and transcription. Targeted modification of several viral components, such as the viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for conducting the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is time consuming and expensive. It is solely dependent on the presence of unique restriction enzyme sites that may or may not be present in the target site. Clustered regularly interspaced short palindromic repeat (CRISPR) along with an RNA-guided nuclease called Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in various cells and organisms. Thus, we utilized in vitro CRISPR/Cas9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification in the viral genome. We have conducted CRISPR/Cas9-mediated insertional mutagenesis and successfully inserted the RFP (red fluorescent protein) reporter construct into the CAV2 genome. We have also utilized the CRISPR/Cas9 system to conduct wild type gene replacement with our desired ligand in the CAV-2 genome to construct tumor-targeted vectors. Results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. For transcriptional targeting, several previously identified tumor up-regulated promoters, CXCR4, Survivin and TERT along with an E2F modified Canine Adenovirus 2 E1A promoter (EEE), were tested for their expression in various canine tumor cells/tissues by employing RT-qPCR and GFP reporter gene expression levels to measure endogenous and exogenous promoter activity, respectively. Endogenous expression levels measured for cTERT, cSurvivin, and cCXCR4 were low for all three, with some non-malignant and some tumor cell lines and tissues expressing the gene. Expression levels from exogenously supplied promoters were measured by both the number of cells expressing the construct and the intensity of expression in individual cells. Exogenously supplied promoters were active in more cells in all tumor lines than in normal cells, with the EEE promoter being most active, followed by cTERT. The intensity of expression varied more with cell type than with specific promoters. Ultimately, no single promoter was identified that would result in reliable expression, regardless of the tumor type. Thus, these findings imply that the three investigated promoters are unsuitable for use as lymphoma-specific promoters. In addition, this data raises the concern that endogenous expression analysis may not accurately predict exogenous promoter activity. We hope that these approaches and findings will help us to construct oncolytic CAV-2 suitable to eliminate current obstacles for oncolytic virotherapy in multicentric canine tumors such as lymphoma. In addition, these approaches may be translated to human patients and will allow adenoviruses to be successfully utilized as gene therapy vectors for a wide range of autoimmune and other diseases beyond hematopoietic malignancies.