Figure 5.

AAV-Vector-System-Mediated Targeted Gene Therapy Based on Post-transcriptional Action of miRNA199a

To investigate the possibility of vector-mediated delivery of a transgene harboring miR199a binding site, AAV8 harboring GLuc as a reporter and cytosine deaminase (CD) as a therapeutic gene was constructed with or without miR199a binding sites at the 3′ UTR of the transgenes, and cells were transduced at an MOI of 100,000 vgs per cell. (A) Construction of plasmid harboring GLuc is indicated, as well as CD flanked by self-complementary inverted terminal repeats of the AAV. (B) Reporter expression after delivery with AAV: both miRNA199a-positive and -negative cells were transduced with scAAV8-GLuc and scAAV8-GLuc-miR199a*3, and the amount of secreted GLuc was quantified. Relative expression of GLuc after transduction with scAAV8-GLuc-miR199a*3 was reported as a percentage of that after transduction with scAAV8-GLuc. (C) Targeted GDEPT after AAV-mediated suicide gene therapy: cells were transduced with scAAV8-CD and scAAV8-CD-miR199a*3 and incubated with the prodrug 5-FC. The percent proliferation was then calculated for scAAV8-CD-miR199a*3 and represented as a percentage of that for scAAV8-CD for each cell type. The significant difference between groups was tested by two-tailed t test using GraphPad Prism v7.0 (data are reported as mean ± SD, n > 3; *p < 0.05).