Projects

1. Construction of tumor specific replicating viruses
   
   
2. Investigation of cell death mechanisms in oncolytic virotherapy
   
   
3. Re-targeting of viral infection
   
   
4. Antitumoral immune response by tumor specific replicating viruses

For the generation of tumor specific replicating adenoviruses we adopted three different strategies.

1.1.Transcriptional control of essential viral genes by the human telomerase-promotor.

The catalytic component of human telomerase reverse transcriptase (hTERT) is not expressed in most primary somatic human cells, whereas the majority of cancer cells reactivate telomerase by transcriptional upregulation of hTERT. We constructed a TSRV by replacing the natural E1A promoter of the adenovirus (Ad wild typ serotype 5) with a 255-bp human TERT-promoter fragment (Wirth et al. 2003). Additionally, an internal ribosomal entry site-enhanced green fluorescent protein cassette was inserted downstream of the E1B locus to monitor viral replication in vivo. Adenoviral replication of hTERT-Ad and enhancement of enhanced green fluorescent protein expression could be observed in all investigated telomerase-positive tumor cell lines. In contrast, hTERT-Ad infection of telomerase-negative primary human hepatocytes did not result in significant replication. The capability of hTERT-Ad to induce cytopathic effects in tumor cells was significantly higher compared with ONYX-015 (an E1B mutated oncolytic virus), regardless of the p53 status of the tumor cells. Single application of low-dose hTERT-Ad to tumor xenografts led to significant inhibition of tumor growth, confirming the potential therapeutic value of conditionally replicative adenoviral vectors.

1.2. Tumor specific viral replication by p53-dependent expression of transcriptional repressors.

The majority of human carcinomas harbor genetic lesions or epigenetic alterations that result in a loss of p53 expression or transcriptional functions. In our laboratory we developed a p53-conditionally replicating adenovirus, designated “Adp53sensor” for oncolytic treatment of p53-dysfunctional tumors. Conditional replication was accomplished by p53-dependent expression of the strong transcriptional repressor GAL4-KRAB, directed against transcription of the adenoviral E1A gene. In p53-dysfunctional cells, Adp53sensor showed strong lytic and replicative properties equivalent to a genetically similar, but p53-independent control vector. In contrast, replication of Adp53sensor was selectively attenuated in cells with functional p53, as demonstrated in various p53-active cell lines, in primary human hepatocytes, and also by systemic infection of p53-normal and p53-knockout mice, respectively. Therapeutic efficacy in vivo was improved compared to the oncolytic virus Onyx-015, an E1B-deleted adenovirus mutant. Remarkably, we found no evidence that p53-analogous transcriptional activity that could be ascribed to other p53-family members could compromise tumor specific replication and therapeutic efficiency of Adp53sensor (Kühnel et al. in press).

1.3. Tumor specific viral replication by p53-dependent RNA interference.

RNA-interference (RNAi) is an evolutionary conserved mechanism of eukaryotic gene regulation and a potent tool for specific gene silencing. In our previous work we could demonstrate that engineered RNAi-networks can be used to manipulate the tropism of viruses by tissue-specific interference with viral replication. We developed an RNAi-controlled adenovirus for conditional replication in p53-dysfunctional tumor cells, since p53-dysfunction represents a preferential target for antineoplastic interventions (Gürlevik et al. 2009). Replication control of this recombinant virus is achieved by p53-selective expression of a self-inhibitory RNAi network consisting of multiple antiviral microRNA transcripts. In cells with transcriptionally active p53, RNAi-mediated knockdown of essential adenoviral genes significantly attenuated viral replication compared to a control virus expressing a scrambled RNAi-network. Attenuated replication was a result of antiviral RNAi since both viruses replicated equivalently in p53-dysfunctional tumor cells. In therapeutic settings in vivo, we could show that RNAi-mediated control of adenoviral replication drastically reduced intrahepatic load of viral DNA compared to the scrambled virus. In contrast, DNA of both viruses accumulated similarly in the livers of p53-knockout mice. Addressing the therapeutic applicability we demonstrate that the recombinant virus efficiently lysed p53-dysfunctional tumors in vitro and in vivo. Inhibitory RNAi-networks to control viral replication can be applied to all transcriptionally regulated DNA viruses and thus provide means to generate specifically replicating vectors for clinical applications.

Adenoviral infection of normal tissue during oncolytic virotherapy can lead to life-threatening toxicities in mouse tumor models. In addition it is well known that the nature of cell death determines the immune response against virus and intracellular antigens. Thus understanding of the molecular mechanisms of virus-induced cell death is important to prevent virotherapy-related toxicities and to optimize virotherapy-induced antitumoral immune responses. In previous work we investigated the molecular mechanisms of extrinsic and intrinsic apoptosis and autophagy in adenovirus-induced cell death (Zender et al. 2003, Mundt et al. 2003, Mundt et al. 2005, Zender et al. 2005, Ito et al. 2006). Adenoviral hepatitis resulted in up-regulation of several pro- and antiapoptotic mediators in the extrinsic and intrinsic death pathway, but triggering of death receptors appears to be essential for adenovirus-mediated cell death of hepatocytes. To prevent liver toxicity during virotherapy we performed the first successful therapeutic application of RNAi in an animal model (Zender et al. 2003). In further experiments we could demonstrate that telomerase-dependent virotherapy overcomes the resistance of HCC against TRAIL and chemotherapy (Wirth et al. 2005). The synergistic effects are explained by a strong down-regulation of Mcl-1 expression through hTERT-Ad that sensitizes HCC for TRAIL and chemotherapy-mediated apoptosis. We observed similar results in VSV-virotherapy of tumors, indicating that Mcl-1 degradation is a general mechanism for sensitizing tumor cells to apoptosis by viruses (Schache et al. 2009).

Expression of cellular receptors determines viral tropism and limits gene delivery by viral vectors. In our laboratory, we investigated the role of several protein transduction domains (PTD) motifs in adenoviral infection. When physiologically expressed, a PTD from human immunodeficiency virus transactivator of transcription (Tat) did not improve adenoviral infection. We therefore fused PTDs to the ectodomain of the coxsackievirusadenovirus receptor (CARex) to attach PTDs to adenoviral fiber knobs (Kühnel et al. 2004). CARex-Tat and CARex-VP22 allowed efficient adenoviral infection in nonpermissive cells and significantly improved viral uptake rates in permissive cells. Expression of CARex-PTDs led to enhanced lysis of permissive and nonpermissive tumor cells by replicating adenoviruses, indicating that CARex-PTDs are valuable tools to improve the efficacy of oncolytic therapy. Current projects investigate different strategies for tumor-specific retargeting.