Z Gastroenterol 2009; 47 - P3_42
DOI: 10.1055/s-0029-1191899

Generation of a conditionally replicating adenovirus harboring a P53-dependent self-destruction switch for treatment of P53-altered tumors

P Schache 1, A Goez 1, E Gürlevik 1, N Strüver 1, MP Manns 1, S Kubicka 1, F Kühnel 1
  • 1Medizinische Hochschule Hannover, Klinik für Gastroenterologie, Hepatologie und Endokrinologie, Hannover

Background/aims: Conditionally replicating adenoviruses represent a promising approach for the treatment of solid tumors. Actual concepts focus on restricting viral replication to malignant cells but no concept exists that targets the viral vector DNA itself for selective destruction in normal cells. Here, we present an p53-dependent oncolytic adenovirus harboring a nuclease function for selective vector destruction in normal cells.

Methods: We constructed conditionally-replicating adenoviruses expressing the meganuclease I-Sce I under control of a p53-dependent promoter. For targeted vector DNA destruction, the viral early genes (E1A/E1B) were flanked by I-Sce I-cleavage sites. For all vectors, controls were cloned wherein I-Sce I was replaced by EGFP.

Results: First, efficacy of DNA cleavage function of I-Sce I was investigated using an I-Sce I-sensitive dual EGFP/luciferase reporter in HepG2 cells. Luciferase assays showed that the reporter was efficiently cleaved by coexpressed I-Sce I. When controlled by a p53-dependent promoter, I-Sce I expression could be detected in p53-positive HepG2 but not in p53-inactive Huh-7-cells. After vector construction, correct protein expression pattern was confirmed by western blot analysis. The results showed that I-Sce I (or EGFP) was expressed in p53-positive, but not in p53-negative cells. In oncolysis assays of p53 positive cells all I-Sce I-expressing viral vectors displayed a superior selectivity as cell lysis was efficiently inhibited compared to their EGFP-expressing controls. In the p53 mutant cell line Huh-7 all vectors showed comparable replication and cell lysis. Further, a combination of I-Sce I mediated vector destruction and an additional Gal4-KRAB dependent repression of E1A resulted in improvement of selectivity.

Conclusions: We established tightly regulated, conditionally replicating adenoviruses that comprise a vector destruction mechanism for improved safety in virotherapeutic treatment of solid tumors.

I-Sce I - conditionally replicating Adenovirus - p53