Diffuse midline glioma H3K27M mutant (DIPG) is a fatal disease and conveys survival of about one year despite the best combination of therapies. Recurrence after treatment is universal, owing in part to the diffusely infiltrative nature of these tumors, the aggressive patterns of local spread, and intra-tumoral cellular and genetic heterogeneity. An engineered replication-competent retrovirus vector (RRV) leverages the efficient amplification process of the viral life cycle. Since retroviruses achieve genomic integration, gene expression is persistent in tumors, tracking cells even as they migrate or metastasize. The pAC3-yCD2 vector is an RRV specifically engineered to deliver a pro-drug activating “suicide” gene encoding cytosine deaminase, which converts 5-fluorocytosine to 5-fluorouracil. This strategy has shown potential benefit in other cancers, and here we sought to test its applicability to DIPG. However, viral transduction in these cells has not been previously performed, and thus we sought to test the gene transduction efficiency of our RRV in DIPG cell lines in vitro.
Both mutant human and mouse DIPG cell lines were grown in serum-free neurosphere culture. Patient-derived DIPG cells were transduced with green fluorescence protein-labeled RRV. Cloning of mStrawberry fluorescence protein-labeled RRV was unsuccessful, so it could not be transduced into the mouse DIPG cells. Light microscopy and flow cytometry were used to measure GFP-labeled RRV gene transduction efficiency by percentage of fluorescent cells. After confirming successful viral transduction, human DIPG cells were then transduced with pAC3-yCD2.
Patient-derived DIPG cells transduced with GFP-labeled RRV demonstrated green fluorescence under the microscope seven days after transduction. In fourteen days, 94.0% of cells transduced with 0.125 mL (1x) and 0.5 mL (4x) RRV were GFP positive, whereas 90.9% of cells transduced with 0.25 mL (2x) of RRV were GFP positive by flow cytometry. Median fluorescence intensity values (arbitrary units) were 2862, 2802, and 3117 for cells transduced with 0.125 mL (1x), 0.25 mL (2x), 0.5 mL (4x) RRV, respectively.
Our retroviral vector can be used to efficiently transduce gene sequences encoding fluorescence proteins, and thus presumably yCD2, in DIPG cells. RRV encoding yCD2 suicide gene therapy may be an effective means of in situ cytolysis in DIPG. Cell toxicity assays will be performed with 5-fluorocytosine prodrug administration to test for cytolysis and successful yCD2 gene transduction in future studies.