Inducible Gene Expression in Tumors Colonized by Modified Oncolytic Vaccinia Virus Strains

Abstract

Exogenous gene induction of therapeutic, diagnostic, and safety mechanisms could be a considerable improvement in oncolytic virotherapy. Here, we introduced a doxycycline-inducible promoter system (comprised of a tetracycline repressor, several promoter constructs, and a tet operator sequence) into oncolytic recombinant vaccinia viruses (rVACV), which were further characterized in detail. Experiments in cell cultures as well as in tumor-bearing mice were analyzed to determine the role of the inducible-system components. To accomplish this, we took advantage of the optical reporter construct, which resulted in the production of click-beetle luciferase as well as a red fluorescent protein. The results indicated that each of the system components could be used to optimize the induction rates and had an influence on the background expression levels. Depending on the given gene to be induced in rVACV-colonized tumors of patients, we discuss the doxycycline-inducible promoter system adjustment and further optimization. IMPORTANCEOncolytic virotherapy of cancer can greatly benefit from the expression of heterologous genes. It is reasonable that some of those heterologous gene products could have detrimental effects either on the cancer patient or on the oncolytic virus itself if they are expressed at the wrong time or if the expression levels are too high. Therefore, exogenous control of gene expression levels by administration of a nontoxic inducer will have positive effects on the safety as well as the therapeutic outcome of oncolytic virotherapy. In addition, it paves the way for the introduction of new therapeutic genes into the genome of oncolytic viruses that could not have been tested otherwise. Cancer remains one of the major health problems worldwide, and novel therapies are direly needed. Over the past decade, therapies which take advantage of tumor-colonizing microorganisms have gained more and more attention and become a significant field of research followed by ongoing clinical trials. Tumorcolonizing microorganisms include pathogenic, nonpathogenic, and even probiotic bacteria, as well as different virus strains. All of those microorganisms have in common the fact that they colonize and replicate in solid tumors and metastases to a much higher extent than in healthy tissues, resulting in tumor-to-organ ratios that by far exceed any other targeted therapies that are based on small molecules and therapeutic antibodies. This phenomenon of tumor-specific enrichment and amplification is either a consequence of rational genetic engineering or based on natural traits of those microorganisms or both. Whatever the basis for the tumorspecific replication is, tumor-colonizing microorganisms are now being used to (over)express heterologous therapeutic proteins within tumor tissues, thereby enhancing the therapeutic efficacy.Some of those therapeutic proteins can be toxic to the patient or even to the microorganism that produces the protein. Therefore, it would be beneficial to exogenously regulate their gene ...

Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent

“…For induction of melanin production, we inserted the tet -regulated gene expression system components into the rVACV genome 15 . The system can be induced with doxycycline, a tetracycline derivate 21 .…”

“…The tyrosinase-negative strains GLV-1h68 14 and GLV-1h312 15 were used as non-melanogenic control viruses. The strain GLV-1h460 encodes the main melanogenic enzyme mTyr under control of the synthetic late promoter followed by the tet-operator sequence tetO .…”

Thyroid hormone and vitamin D regulate VGF expression and promoter activity

“…First, the ZsGreen gene present in the promoterless pZsGreen1-1 vector (Clontech Laboratory) was replaced with CBG992AmRFP from pCR2CBG992AmRFP (pRFP, Stritzker et al . 2014 ).…”

Oncolytic virotherapy evolved into the fourth generation as tumor immunotherapy

“…Jochen Stritzker et al . has characterized a doxycycline-inducible promoter system in oVVs, which was proven to be beneficial to OVT [ 123 ]. Therefore, determination of the structure and characteristics of various viruses and tumor cells will be greatly beneficial for the development of efficient OVT.…”