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

Francis Rousset,Patrick Salmon,Simon Bredl,Ophélie Cherpin,Marta Coelho,Renier Myburgh,Marco Alessandrini,Michael Perny,Marta Roccio,Roberto F Speck,Pascal Senn,Karl Heinz Krause

doi:10.1016/j.omtn.2018.12.004

Francis Rousset, Patrick Salmon + Show 10 more

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https://doi.org/10.1016/j.omtn.2018.12.004

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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.

Highlights

The discovery and characterization of microRNA genes and their regulatory mechanisms provided a novel understanding of physiological regulation of gene expression,[1] and opened new possibilities for miRNA-based therapeutics.[2]
The pathway has not lived up to its therapeutic potential.[2,6] small interfering RNAs (siRNAs) are most advanced in clinics[6]; they are short-lived in vivo, and their transient effect would require repeated in vivo delivery for efficient long-term gene correction.[7] short hairpin RNAs (shRNAs), which bypass DROSHA processing, may overload the cytoplasm with double-stranded RNA and lead to toxicity by obstructing the natural miRNA pathway.[8,9]
With the spacer between the promoter and the miRNA, an efficient CCR5 knockdown was observed with a single hairpin, which was markedly enhanced with a concatenated triple hairpin construct

Summary

Introduction

The discovery and characterization of microRNA (miRNA) genes and their regulatory mechanisms provided a novel understanding of physiological regulation of gene expression,[1] and opened new possibilities for miRNA-based therapeutics.[2]. The pathway has not lived up to its therapeutic potential.[2,6] siRNAs are most advanced in clinics[6]; they are short-lived in vivo, and their transient effect would require repeated in vivo delivery for efficient long-term gene correction.[7] shRNAs, which bypass DROSHA processing, may overload the cytoplasm with double-stranded RNA and lead to toxicity by obstructing the natural miRNA pathway.[8,9] Synthetic miRNAs mimic the natural pathway and should overcome the above limitations,[10] but their use might be limited because of a relatively weak knockdown activity of miRNA, as compared with shRNAs.[11]

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