Figure 4.

Optimization of the miH12 expression cassette to address efficacy and passenger strand activity. (a) Schematic representation of the pri-miH12 expression cassette with the CMV immediate-early enhancer fused to chicken β-actin (CAG) promoter and human growth hormone polyadenylation (hGH polyA) signal. (b) Ten cellular pri-miRNA scaffolds were selected from miRBase database (www.mirbase.org) based on the guide/passenger strand prevalence and RNA folding. Twenty-one nt of the guide strand were replaced by the mature miH12 sequence and the passenger strand was corrected in order to preserve pri-miH12 scaffolding. Based on miRBase, the predicted guide and passenger strand sequences of the cellular pre-miRNA scaffolds are indicated in red and blue, respectively. (c) LucHTT(mt) reporter knock-down by ten miH12 scaffolds. Human embryonic kidney (HEK)293T cells were cotransfected with 100 ng LucHTT(mt) reporter and 10 ng miH12 constructs. Renilla (RL) and firefly (FL) luciferases were measured 2 days post-transfection and RL was normalized to FL expression. Scrambled (Ctrl) served as a negative control and was set at 100%. (d) Titration curve of miH12 embedded in the human miR-1, miR-101, miR-122, miR-135, miR-203, and miR-451 scaffolds. HEK293T cells were cotransfected with 50 ng LucHTT(mt) reporter and 1, 10, 50, 100, or 250 ng of miH12 constructs. Luciferase expression was measured as described in (c). (e) In vitro passenger strand activity of miH12-1, miH12-26, miH12-101, miH12-122, miH12-135, miH12-155, miH12-203, miH12-335, and miH12-451 scaffolds. For the LucPassenger reporters, reverse complement sequences to the miH12 passenger strand sequences were cloned behind RL. In addition, FL was coexpressed from the vector as an internal control. Transfections and luciferase measurements were performed as in (c). The luciferase knock-down are representative figures of three independent experiments.