ABSTRACT
Dumbbell-shaped DNA minimal vectors represent genetic vectors solely composed of the gene expression cassette of interest and terminal closing loop structures. Dumbbell vectors for small hairpin RNA or microRNA expression are extremely small-sized, which is advantageous with regard to cellular delivery and nuclear diffusion. Conventional strategies for the generation of small RNA-expressing dumbbell vectors require cloning of a respective plasmid vector, which is subsequently used for dumbbell production. Here, we present a novel cloning-free method for the generation of small RNA-expressing dumbbell vectors that also does not require any restriction endonucleases. This new PCR-based method uses a universal DNA template comprising an inverted repeat of the minimal H1 promoter and the miR-30 stem. The sequences coding for small RNA expression are introduced by the PCR primers. Dumbbells are formed by denaturing and reannealing of the PCR product and are covalently closed using ssDNA ligase. The new protocol generates plus- and/or minus-strand dumbbells, both of which were shown to trigger efficient target gene knockdown. This method enables fast, cheap production of small RNA-expressing dumbbell vectors in a high throughput-compatible manner for functional genomics screens or, as dumbbells are not prone to transgene silencing, for knockdown studies in primary cells.
ABSTRACT
Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.
