Diphtheria Toxin Resistance Selective Marker for in vivo selection

Personalized medicine is a medical procedure that is based upon a patient's unique genetic makeup. Researchers use this information to find prevention, screening and treatment strategies that are effective. By performing these tests on cancer cells, scientists may be able to customize treatment to each patient's needs. One of the most efficient ways to test a specific patient's tumor response to various available treatments is to implant small pieces of their tumor in a mouse host. Growth (or lack thereof) of the tumor is an indication of the failure or success of the treatment for that particular tumor. However, assessing growth of implanted human tumors in mice is difficult, and requires invasive and time‐consuming techniques. Therefore, in this study, we have developed a novel tool; a lentivirus called NIGHT‐OWL ( N on‐ I nvasive G rowth of H uman T umors‐ O n W ith the L uciferase enzyme). NIGHT‐OWL can be applied directly to patient‐derived xenografts (PDX) and selected for in vivo . After selection, tumor cells in the PDX will secrete luciferase, which can be detected in bodily fluids such as urine. Thus, development of this tool addresses a critical barrier in the field and allows non‐invasive assessment of the PDX response to treatment. First, the purpose of the study was to construct a lentiviral vector containing a selective marker providing human cancer cells resistance to diphtheria toxin (DT). DT can then be applied for in vivo selection of human cancer cells in a patient derived xenograft (PDX) grown in a rodent host. The vector also encodes a secreted luciferase, Metridia Luciferase cDNA (mLUC), which allows for measurements of tumor growth. In addition, the vector also expresses a red fluorescent protein which provides the ability to take live images to visually see the growth of the tumor over time. To provide resistance to DT, the vector consists of a short hairpin RNA sequence, silencing the DPH2 gene, an enzyme that catalyzes a key step in diphthamide biosynthesis, a histidine modification process known to occur only on His715 of eukaryotic elongation factor 2 (EEF2). Diphtheria toxin inhibits EEF2 by catalyzing the transfer of NAD+ to diphthamide, and as a result, in the absence of DPH2, His715 is not converted to diphthamide and the cell is in‐sensitive to diphtheria toxin. The lentiviral vector was transfected into HEK 293 lentiviral packaging cell line and viral particles were collected. The viral particles were then used for stable transduction into a human cancer cell line. Further tests were taken to see whether the cells were expressing the luciferase enzyme, the resistance against diphtheria toxin and if the cells were also expressing the red fluorescence protein. In conclusion, we have developed a novel tool that can inexpensively and non‐invasively provide monitoring of human PDX tumor responses to different therapeutic approaches thereby facilitating a major advance in research approaches for individualized medicine. Support or Funding Information Program supported by the Cancer Therapy and Research Cancer at UT Health, P30 CA054174‐Continuing Umbrella of Research Experience (CURE) Grant P30 CA054174‐22S2 CRTC PROSTATE SPORE PILOT AWARD This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .