Optimizing Synthetic miRNA Minigene Architecture for Efficient miRNA Hairpin Concatenation and Multi-target Gene Knockdown

Abstract

Synthetic microRNA (miRNA) minigenes (SMIGs) have a major potential for molecular therapy; however, their optimal architecture still needs to be determined. We have previously optimized the stem structure of miRNA hairpins for efficient gene knockdown. Here, we investigate the overall architecture of SMIGs driven by polymerase II-dependent promoters. When miRNA hairpins were placed directly behind the promoter, gene knockdown was inefficient as compared with constructs containing an intercalated sequence (“spacer”). Spacer sequence was relevant for knockdown efficiency and concatenation potential: GFP-based sequences (even when truncated or including stop codons) were particularly efficient. In contrast, a spacer of similar length based on a CD4 intronic sequence was entirely inefficient. Spacer sequences influenced miRNA steady-state levels without affecting transcript stability. We demonstrate that with an optimized spacer, up to five concatenated hairpins targeting two different genes are efficiently expressed and able to knock down their respective targets. Transplantation of hematopoietic stem cells containing a CCR5 knockdown SMIG demonstrated a sustained in vivo efficacy of our approach. In summary, we have defined features that optimize SMIG efficiency. Based on these results, optimized knockdown of genes of interest, such as the HIV co-receptor CCR5 and the NADPH oxidase subunit p22phox, was achieved.

NADPH Oxidase 3 Deficiency Protects From Noise-Induced Sensorineural Hearing Loss

“…Whether the commonly used glucocorticoids for these situations today work through inhibition of NOX3, or whether a more specific NOX3 inhibition through pharmacological or molecular intervention is more effective remains to be investigated in the future. In absence of a bona fide NOX3 inhibitor, a nucleotide-based approach seems to be more promising ( Rousset et al, 2019 ), at least based on the situation today.…”

“…Based on these findings, an active role of the ROS generating enzyme NOX3 in the pathophysiology of noise-induced hearing loss can be demonstrated, in line with NOX3 implication in other forms of acquired hearing loss such as age-related hearing loss and cisplatin-induced hearing loss. The exclusive and specific expression of NOX3 in the inner ear opens interesting opportunities for pharmacological ( Augsburger et al, 2019 ) or molecular ( Rousset et al, 2019 ) interventions aiming at prevention of several acquired forms of hearing loss including noise-induced hearing loss.…”

Arylsulfatases and neuraminidases modulate engagement of CCR5 by chemokines by removing key electrostatic interactions

“…The microRNA-based lentivector (mirGE) inducible knockdown system 26 – 28 was used to target each of the 8 ARS candidates. An automated scoring system 29 was used to identify sequence stretches in each target ARS mRNA and design appropriate mirGE DNA sequences. Synthesized target DNA (Thermo Fisher Scientific) was cloned into an acceptor vector (pENTR) by ligation and then transferred by gateway cloning into an autoinducible lentivector plasmid (pCLX) as previously described 28 .…”

Stable overexpression of native and artificial miRNAs for the production of differentially fucosylated antibodies in CHO cells

“…Different expression levels of miRNAs from plasmids containing a spacer between the promoter and the miRNA were already observed previously. Consequently, using GFP as a rather long spacer sequence similar to the pcDNA6.2 plasmid, the strongest effect of miRNA-mediated regulation was observed [60].…”