The purpose of this project is to determine whether KM110r is an effective and safe oncolytic virus for cancer therapy.

Specifically this project attempts to:

• Assess the selectivity of KM110r by exploring its effect on cancerous bone (osteosarcoma) cells (U2OS) and normal pre-cursor bone (osteoblast) cells (hFOB)
• Determine whether induced genetic differentiation alters the permissiveness of hFOB cells to KM110r
• Isolate the stage during cell differentiation at which the hFOB cells respond to KM110r infection if KM110r responds to normal changes during differentiation
• Determine why hFOB cells are able to ignore KM110r
• Determine why U2OS cells are preferentially susceptible (permissive) to the virus

The selectivity of KM110r will be dependent on its cytotoxicity towards U2OS osteosarcoma cells and its capability to ignore hFOB osteoblast cells. KM110r will be considered selective if it demonstrates cytotoxicity towards U2OS osteosarcoma cells while remaining non-toxic to hFOB osteoblast cells. Since the desired effect of cancer therapies is to successfully eradicate all cancerous cells while ignoring non-cancerous cells, the possible efficacy of KM110r may lead to its classification as a selectively oncolytic virus therapy.

hFOB cells are engineered with a temperature-sensitive mutation (tsA58) that drives differentiation in response to temperature change. Thus by varying the incubation temperatures of hFOB cell lines, normal genetic differentiation of the progenitor (hFOB) cells will occur. If a point can be identified during in vitro cell differentiation at which the hFOB cells respond to viral infection, the understanding of which genetic changes permit oncolytic virus infection will be enhanced. This would lead to identifying the point along the differentiation pathway that would maximize oncolytic virus treatment efficacy.  Depending on the results of hFOB infection, Km110r will either be a safe oncolytic virus or a useful tool to study normal differentiation.

The sequence of normal genetic changes that occur during differentiation of precursor (stem cells or hFOB cells) into differentiated cells will be simulated by using various temperature changes. The cells will then be analyzed to determine whether differentiation alters the susceptibility of the hFOB cells to KM110r infection. If so, then the differences between the genetic makeup of the normal undifferentiated hFOB cell and that of the genetically altered cell at the point at which it responds to the viral infection will be investigated with the intent of determining why the virus is unable to infect normal cells, yet destroys the genetically altered cells.

Although it is known that KM110r successfully induces lysis in cancerous cells and is not toxic to hFOB cells, the efficacy of KM110r has never been tested on hFOB or U2OS cells over the course of various temperatures. KM110r has only ever been tested on undifferentiated progenitor cell lines, and so this is the first time that the selectivity of KM110r will be investigated during differentiation. This is the first time that KM110r has been tested on U2OS cells at various incubation temperatures. This experimentation will be the first of its kind ever performed in the world.