title: Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression.
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.
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Xiaoou
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Han
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Cui Rong
authors.initials: CR
authors.lastname: Teo
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Genim Siu Xian
authors.initials: GS
authors.lastname: Tan
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Sok Chin
authors.initials: SC
authors.lastname: Goh
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.|Department of Cell and Systems Biology, University of Toronto, Ramsay Wright Laboratories, Toronto, Ontario, Canada.
authors.forename: Parasvi
authors.initials: P
authors.lastname: Patel
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Yiqiang Kevin
authors.initials: YK
authors.lastname: Chua
authors.affiliation: Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, Singapore.
authors.forename: Nasirah Banu Sahul
authors.initials: NB
authors.lastname: Hameed
authors.affiliation: Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, Singapore.
authors.forename: Antonio
authors.initials: A
authors.lastname: Bertoletti
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Volker
authors.initials: V
authors.lastname: Patzel
mesh_terms: D002460:Cell Line; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007438:Introns; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics; D022661:RNA, Untranslated / Q000737:chemistry / Q000235:genetics*; D013601:T-Lymphocytes
chemical_list: D012322:RNA Precursors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D022661:RNA, Untranslated