Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression

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 impro...

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Bibliographic Details
Published in:Molecular therapy Vol. 24; no. 9; pp. 1581 - 1591
Main Authors: Jiang, Xiaoou, Yu, Han, Teo, Cui Rong, Tan, Genim Siu Xian, Goh, Sok Chin, Patel, Parasvi, Chua, Yiqiang Kevin, Hameed, Nasirah Banu Sahul, Bertoletti, Antonio, Patzel, Volker
Format: Journal Article
Language:English
Published: United States Elsevier Inc 01-09-2016
Elsevier Limited
Nature Publishing Group
Subjects:
Adenosine
Antibiotics
Cell Line
Design
Drug resistance
Enhancer Elements, Genetic
Gene Expression
Gene Knockdown Techniques
Gene Targeting
Genetic Vectors - genetics
Humans
Immunology
Introns
MicroRNAs
Nucleic Acid Conformation
Original
Peptides
Plasmids
Plasmids - genetics
RNA Precursors - chemistry
RNA Precursors - genetics
RNA Splicing
RNA, Messenger - chemistry
RNA, Messenger - genetics
RNA, Small Interfering - chemistry
RNA, Small Interfering - genetics
RNA, Untranslated - chemistry
RNA, Untranslated - genetics
T-Lymphocytes
Toxicity
Vectors (Biology)
Singapore
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Summary: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.
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ISSN:1525-0016
1525-0024
DOI:10.1038/mt.2016.138