Abstract
Multiple types of oncolytic viruses are currently under investigation in clinical trials. To optimize therapeutic outcomes it is believed that the plethora of different tumor types will require a diversity of different virus types. Sendai virus (SeV), a murine parainfluenza virus, displays a broad host range, enters cells within minutes and already has been applied safely as a gene transfer vector in gene therapy patients. However, SeV spreading naturally is abrogated in human cells due to a lack of virus activating proteases. To enable oncolytic applications of SeV we here engineered a set of novel recombinant vectors by a two-step approach: (i) introduction of an ubiquitously recognized cleavage-motive into SeV fusion protein now enabling continuous spreading in human tissues, and (ii) profound attenuation of these rSeV by the knockout of viral immune modulating accessory proteins. When employing human hepatoma cell lines, newly generated SeV variants now reached high titers and induced a profound tumor cell lysis. In contrast, virus release from untransformed human fibroblasts or primary human hepatocytes was found to be reduced by about three log steps in a time course experiment which enables the cumulation of kinetic differences of the distinct phases of viral replication such as primary target cell infection, target cell replication, and progeny virus particle release. In a hepatoma xenograft animal model we found a tumor-specific spreading of our novel recombinant SeV vectors without evidence of biodistribution into non-malignant tissues. In conclusion, we successfully developed novel tumor-selective oncolytic rSeV vectors, constituting a new tool for virotherapy of solid tumors being ready for further preclinical and clinical development to address distinct tumor types.
Highlights
One of the most important barriers that limit the successful treatment of cancer today is constituted by the presence of primary or by the development of secondary resistance phenomena
Generation of recombinant Sendai viruses For the generation of SeV particles that conditionally replicate in tumor cells and thereby induce a profound oncolysis, a twostep-approach was performed: first, the natural protease cleavagesite of SeV-F protein was replaced by a cleavage-site which can be cleaved by ubiquitously available proteases followed by an artificial attenuation of SeV that selectively exploits tumor cell defects within the innate defense system
To further investigate the cytotoxic and especially the cytolytic potential in tumor cells we examined the cellular viability cell mass determined by a sulforhodamine B (SRB) assay as well as the loss of membrane integrity by determination of lactate dehydrogenase (LDH) release into the cellular supernatant 72 hpi
Summary
One of the most important barriers that limit the successful treatment of cancer today is constituted by the presence of primary or by the development of secondary resistance phenomena. Several different mechanisms leading to a virusinduced, cancer cell-specific killing have been found, such as activation of viral particles by cancer-specific proteases, entry through cancer cell-specific receptors or exploiting specific defects of cancer cells [5]. An example for such tumor cell defect is the inability of most tumor cells to produce or to respond to interferon (IFN) after viral infection [6,7,8]. Due to the diversity of the so far established viral systems and the plethora of different tumor types that have to be addressed, it is challenging to identify or develop distinct oncolytic viruses that are most suitable for a subset of tumor entities
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