A novel Adenoviral miRNA, a candidate for development of a novel gene therapy startegy

In 2017, a novel miRNA was found at the MLTU-region of adenoviral genome, termed as MLP-TSS-sRNA. This current study started with performing a series of mutations in the MLP-TSS-sRNA in order to investigate how the MLP-TSS-sRNA as a single stranded small RNA was protected from rapid RNA degradation in transfected cells (in vivo). Since the hairpin structure of this small RNA was considered to be the reason to its high stability, the deletions of nucleotides were occurred inside the complementary region and the loop of the hairpin structure. Three variants of MLP-TSS-sRNAs were therefore transfected into the A549-lung epithelial cancer cell line and measured during times series studies. The results showed that the wild type form of this small RNA has the highest stability. Subsequently, a panel of different synthetic single-stranded RNAs, in which the MLP-TSS-sRNA sequence was modified to target different genes of interest, was used to compare its suppressive efficiency to the more traditional double stranded small interfering RNA “siRNA” or miRNA mimics. To this, the MLP-TSS-sRNA sequence was modified in such a way that it targeted the Dicer mRNA, thus termed as 3s-dicer-miRNA. Successful suppression of the Dicer mRNA as a consequence of using this modified 3s-dicer-miRNA sequence could emphasize that, theoretically, any possible mRNA of interest could be targeted. To express this miRNA inside a host cell, its sequence was incorporated in a CMV-driven plasmid vector system, upstream of the gene encoding for the HDV-ribozyme, which showed to be functional in vitro, but not in vivo. On the other hand, the vector system showed a clear tendency of being functional even in vivo, once it was put into the test by co-transfecting it with a Dicer plasmid inside 293-cells