112. A Novel Oncolytic Adenovirus Based on Human Adenovirus Serotype 17

Abstract

Most existing oncolytic adenoviruses (AdV) are based on human AdV type 5 from species C (hAdV-C5). Clinical efficacy of hAdV-C5 based oncolytic viruses is limited by variable expression levels of coxsackie- and adenovirus receptor (CAR) in different tumour cells and insufficient replication rates. Additionally, patients exposed to AdV develop serotype specific neutralizing antibodies compromising reapplication. Thus, development of novel oncolytic AdV based on other serotypes may help overcoming these limitations. It may additionally allow repeated dosing in humans and add to the repertoire of antitumour agents in general. We further hypothesised that oncolytic efficacy of the candidate virus can be augmented by expression of RNAi suppressor protein P19 (Lecellier et al., Nature 2005) as has been shown previously for hAdV-C5 (Rauschhuber et al, Sci. Rep. 2013). Based on a computational model AdV of species D including type 17 (hAdV-D17) are predicted to have the highest probability of expressing pre-miRNA compared to other group AdV (Pfeffer et al, Nat. Methods 2005). Moreover, high infection efficiency of EA.hy926 cells, a vascular endothelium tumor cell line that lacks CAR (Jing Liu, unpublished) with AdV17 as well as absence of cross-reactivity with AdVC5 may allow exploitation for AdV-C5-insensitive tumors and systemic therapy. Here we aim at evaluating a novel hAdV-D17-based, p19-containing oncolytic AdV as novel candidate for oncolytic applications in different major tumour cell lines and for toxicity in non-malignant cells. Thus, we cloned hAdV-17 and a P19-containing hAdV-D17 based virus by a novel seamless recombineering technique (Zhang et al, unpublished). In order to allow P19 expression from the adenoviral vector genome, the P19 cDNA was fused via an internal ribosome entry site (IRES) to the late fiber gene. To obtain control viruses, GFP was fused via an IRES to the late fiber gene. After release of the respective recombinant adenoviral genomes from plasmids containing the complete DNA molecule, linearized viral DNA was transfected into A549 cells to perform rescue experiments. To evaluate oncolytic potential we are currently comparing it with wild-type hAdV-D17 and hAdV-C5 using common oncolytic essays. Evaluation in primary tumour cells and eventually in an animal model will allow making assumptions about feasibility of oncolytic virotherapy with this vector. In total we believe that HAdV17-based vectors hold promise for oncolytic applications and may be improved by RNAi-suppression.