Engineering Periodic shRNA for Enhanced Silencing Efficacy

Abstract

RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.

Figure 1

Design of open-ended periodic shRNA. (a) Schematic of the synthesis of open-ended p-shRNA by molecular engineering of p-shRNA structures enzymatically assembled from a dumbbell DNA template. (b,c) Analysis of RNase T1 digestion (1 hour at 37 °C) of p-shRNA synthesized from the template in a (p-sh25) by 15% Tris-borate-EDTA (TBE) native polyacrylamide gel electrophoresis (PAGE) (b) and 15% TBE-Urea denaturing PAGE (c), compared to a standard 63 nt RNA of the same sequence as the predicted product.

Figure 2

Effects of template loop size and sequence on p-shRNA folding. (a) Schematic of the effect of transcription initiation site on the relative positions of the G-containing and non-G-containing loops in p-shRNA and the resulting main RNase T1 digestion products. (b) DNA templates with varying loop sizes and sequences. Green circles indicate loops at which T7 RNA polymerase initiates transcription. (c) Analysis of p-shRNA structures generated from templates in a, before and after RNase T1 treatment for 1 hour at 37 °C, by 15% TBE native polyacrylamide gel electrophoresis, with the main RNase T1 digestion products depicted.

Figure 3

Comparison of Dicer processing efficiencies for p-shRNA and op-shRNA. Analysis by 15% native polyacrylamide gel electrophoresis of p-sh25 and op-sh25 following incubation with recombinant human Dicer at 37 °C for up to 24 hours, each shown along with a control incubated for 24 hours without Dicer, and double-stranded RNA markers (M).

Figure 4

Comparison of p-shRNA, op-shRNA, and siRNA transfection efficiencies in various cancer cell lines. (a,b) Green fluorescent protein (GFP) knockdown in GFP-expressing HeLa cells by GFP-targeting and scrambled (scr) p-sh25, op-sh25, and siRNA transfected with Lipofectamine 2000 (a) and TransIT-X2 (b), with the indicated p-sh25 and op-sh25 concentrations based on effective siRNA repeats. (c) GFP knockdown in HeLa-GFP cells by op-shRNAs with 21, 25, 27, and 29 bp stems, using TransIT-X2. (d) Knockdown duration of op-sh25 and siRNA (5 nmol/l) with TransIT-X2 in HeLa-GFP cells. (e,f) Luciferase knockdown in luciferase-expressing SKOV3 (e) and UCI101 (f) cells by luciferase-targeting and scr p-sh25, op-sh25, and siRNA (5 nmol/l) with TransIT-X2. Results are presented as mean ± SEM, n = 3. *P < 0.05, ***P < 0.001.

Figure 5

Evaluation of op-shRNA and siRNA complexation with cationic polymer. (a) Illustration of op-shRNA and siRNA complexation with cationic polymer. (b,c) Agarose gel shift assays of op-shRNA (b) and siRNA (c) complexed with 2 kDa branched polyethyleneimine (PEI) at increasing N/P ratios. (d,e) Dynamic light scattering measurements of the number average hydrodynamic diameters (d) and zeta potentials (e) of op-shRNA and siRNA complexes formed with 2 kDa branched PEI. Results are presented as mean ± SEM, n = 3. (f,g) Heparin displacement assays of op-shRNA (f) and siRNA (g) complexes with 2 kDa branched PEI formed at N/P 15, analyzed by agarose gel electrophoresis.