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 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.

Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression.

Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which are fraught with risk, but dumbbell generation appears to be costly and time-consuming. We developed a new PCR-based method for dumbbell production which comprises only two steps. First, PCR amplification of the therapeutic expression cassette using chemically modified primers to form a ready-to-ligate DNA structure; and second, a highly efficient intramolecular ligation reaction. Compared with conventional strategies, the new method produces dumbbell vectors more rapidly, with higher yields and purity, and at lower costs. In addition, such produced small hairpin RNA expressing dumbbells triggered superior target gene knockdown compared with conventionally produced dumbbells or plasmids. Our novel method is suitable for large-scale dumbbell production and can facilitate clinical applications of this vector system.

A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA.

Several methods for the detection of RNA have been developed over time. For small RNA detection, a stem-loop reverse primer-based protocol relying on TaqMan RT-PCR has been described. This protocol requires an individual specific TaqMan probe for each target RNA and, hence, is highly cost-intensive for experiments with small sample sizes or large numbers of different samples. We describe a universal TaqMan-based probe protocol which can be used to detect any target sequence and demonstrate its applicability for the detection of endogenous as well as artificial eukaryotic and bacterial small RNAs. While the specific and the universal probe-based protocol showed the same sensitivity, the absolute sensitivity of detection was found to be more than 100-fold lower for both than previously reported. In subsequent experiments, we found previously unknown limitations intrinsic to the method affecting its feasibility in determination of mature template RISC incorporation as well as in multiplexing. Both protocols were equally specific in discriminating between correct and incorrect small RNA targets or between mature miRNA and its unprocessed RNA precursor, indicating the stem-loop RT-primer, but not the TaqMan probe, triggers target specificity. The presented universal TaqMan-based RT-PCR protocol represents a cost-efficient method for the detection of small RNAs.

Micro-RNAs in regenerating lungs: an integrative systems biology analysis of murine influenza pneumonia.

BACKGROUND: Tissue regeneration in the lungs is gaining increasing interest as a potential influenza management strategy. In this study, we explored the role of microRNAs, short non-coding RNAs involved in post-transcriptional regulation, during pulmonary regeneration after influenza infection. RESULTS: We profiled miRNA and mRNA expression levels following lung injury and tissue regeneration using a murine influenza pneumonia model. BALB/c mice were infected with a sub-lethal dose of influenza A/PR/8(H1N1) virus, and their lungs were harvested at 7 and 15 days post-infection to evaluate the expression of ~300 miRNAs along with ~36,000 genes using microarrays. A global network was constructed between differentially expressed miRNAs and their potential target genes with particular focus on the pulmonary repair and regeneration processes to elucidate the regulatory role of miRNAs in the lung repair pathways. The miRNA arrays revealed a global down-regulation of miRNAs. TargetScan analyses also revealed specific miRNAs highly involved in targeting relevant gene functions in repair such as miR-290 and miR-505 at 7 dpi; and let-7, miR-21 and miR-30 at 15 dpi. CONCLUSION: The significantly differentially regulated miRNAs are implicated in the activation or suppression of cellular proliferation and stem cell maintenance, which are required during the repair of the damaged lungs. These findings provide opportunities in the development of novel repair strategies in influenza-induced pulmonary injury.

Regulators of mammalian Hippo pathway in cancer.

Hippo pathway, originally discovered in Drosophila, is responsible for organ size control. The pathway is conserved in mammals and has a significant role in restraining cancer development. Regulating the Hippo pathway thus represents a potential therapeutic approach to treat cancer, which however requires deep understanding of the targeted pathway. Despite our limited knowledge on the pathway, there are increasing discoveries of new molecules that regulate and modulate the Hippo downstream signaling particularly in various solid malignancies, from extracellular stimuli or via pathway crosstalk. Herein, we discuss the roles of newly identified and key regulators that connect with core components (MST1/2, LATS1/2, SAV1, and MOB1) and downstream effector (YAP) in the Hippo pathway having an important role in cancer development and progression. Understanding of the mammalian Hippo pathway regulation may shed new insights to allow us selecting the right oncogenic targets and designing effective drugs for cancer treatments.

Formation of Minimised Hairpin Template-transcribing Dumbbell Vectors for Small RNA Expression.

A major barrier for using non-viral vectors for gene therapy is the short duration of transgene expression in postmitotic tissues. Previous studies showed transgene expression from conventional plasmid fell to sub-therapeutic level shortly after delivery even though the vector DNA was retained, suggesting transcription was silenced in vivo ( Nicol et al., 2002 ; Chen et al., 2004 ). Emerging evidence indicates that plasmid bacterial backbone sequences are responsible for the transcriptional repression and this process is independent of CpG methylation ( Chen et al., 2008 ). Dumbbell-shaped DNA vectors consisting solely of essential elements for transgene expression have been developed to circumvent these drawbacks. This novel non-viral vector has been shown to improve transgene expression in vitro and in vivo ( Schakowski et al., 2001 and 2007). Here we describe a novel method for fast and efficient production of minimised small RNA-expressing dumbbell vectors. In brief, the PCR-amplified promoter sequence is ligated to a chemically synthesized hairpin RNA coding DNA template to form the covalently closed dumbbell vector. This new technique may facilitate applications of dumbbell-shaped vectors for preclinical investigation and human gene therapy.

Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors.

Minimal DNA vectors exclusively comprising therapeutically relevant sequences hold great promise for the development of novel therapeutic regimen. Dumbbell-shaped vectors represent non-viral non-integrating DNA minimal vectors which have entered an advanced stage of clinical development ( Hardee et al., 2017 ). Spliceable introns and DNA nuclear import signals such as SV40 enhancer sequences are molecular features that have found multiple applications in plasmid vectors to improve transgene expression. In dumbbells however, effects triggered by introns were not investigated and DNA-based nuclear import sequences have not found applications yet, presumably because dumbbell vectors have continuously been minimized with regard to size. We investigated the effects of an intron and/or SV40 enhancer derived sequences on dumbbell vector driven reporter gene expression. The implementation of a spliceable intron was found to enhance gene expression unconditionally in all investigated cell lines. Conversely, the use of the SV40 enhancer improved gene expression in a cell type-dependent manner. Though both features significantly enlarge dumbbell vector size, neither the intron nor the enhancer or a combination of both revealed a negative effect on gene expression. On the contrary, both features together improved dumbbell-driven gene expression up to 160- or 56-fold compared with plasmids or control dumbbells. Thus, it is highly recommended to consider an intron and the SV40 enhancer for dumbbell vector design. Such an advanced design can facilitate pre-clinical and clinical applications of dumbbell-shaped DNA vectors.

Osteopontin as potential biomarker and therapeutic target in gastric and liver cancers.

Gastric cancer and liver cancer are among the most common malignancies and the leading causes of death worldwide, due to late detection and high recurrence rates. Today, these cancers have a heavy socioeconomic burden, for which a full understanding of their pathophysiological features is warranted to search for promising biomarkers and therapeutic targets. Osteopontin (OPN) is overexpressed in most patients with gastric and liver cancers. Over the past decade, emerging evidence has revealed a correlation of OPN level and clinicopathological features and prognosis in gastric and liver cancers, indicating its potential as an independent prognostic indicator in such patients. Functional studies have verified the potential of OPN knockdown as a therapeutic approach in vitro and in vivo. Furthermore, OPN mediates multifaceted roles in the interaction between cancer cells and the tumor microenvironment, in which many details need further exploration. OPN signaling results in various functions, including prevention of apoptosis, modulation of angiogenesis, malfunction of tumor-associated macrophages, degradation of extracellular matrix, activation of phosphoinositide 3-kinase-Akt and nuclear factor-κB pathways, which lead to tumor formation and progression, particularly in gastric and liver cancers. This editorial aims to review recent findings on alteration in OPN expression and its clinicopathological associations with tumor progression, its potential as a therapeutic target, and putative mechanisms in gastric and liver cancers. Better understanding of the implications of OPN in tumorigenesis might facilitate development of therapeutic regimens to benefit patients with these deadly malignancies.