Differential effects of microRNAs miR-21, miR-99 and miR-145 on lung regeneration and inflammation during recovery from influenza pneumonia.

In a mouse model of influenza pneumonia, we previously documented that proliferating alveolar type II (AT2) cells are the major stem cells involved in early lung recovery. Profiling of microRNAs revealed significant dysregulation of specific ones, including miR-21 and miR-99a. Moreover, miR-145 is known to exhibit antagonism to miR-21. This follow-up study investigated the roles of microRNAs miR-21, miR-99a, and miR-145 in the murine pulmonary regenerative process and inflammation during influenza pneumonia. Inhibition of miR-21 resulted in severe morbidity, and in significantly decreased proliferating AT2 cells due to impaired transition from innate to adaptive immune responses. Knockdown of miR-99a culminated in moderate morbidity, with a significant increase in proliferating AT2 cells that may be linked to PTEN downregulation. In contrast, miR-145 antagonism did not impact morbidity nor the proliferating AT2 cell population, and was associated with downregulation of TNF-alpha, IL1-beta, YM1, and LY6G. Hence, a complex interplay exists between expression of specific miRNAs, lung regeneration, and inflammation during recovery from influenza pneumonia. Inhibition of miR-21 and miR-99a (but not miR-145) can lead to deleterious cellular and molecular effects on pulmonary repair and inflammatory processes during influenza pneumonia.

Non-Covalent Linkage of Helper Functions to Dumbbell-Shaped DNA Vectors for Targeted Delivery.

Covalently closed dumbbell-shaped DNA delivery vectors comprising the double-stranded gene(s) of interest and single-stranded hairpin loops on both ends represent a safe, stable and efficacious alternative to viral and other non-viral DNA-based vector systems. As opposed to plasmids and DNA minicircles, dumbbells can be conjugated via the loops with helper functions for targeted delivery or imaging. Here, we investigated the non-covalent linkage of tri-antennary N-acetylgalactosamine (GalNAc3) or a homodimer of a CD137/4-1BB-binding aptamer (aptCD137-2) to extended dumbbell vector loops via complementary oligonucleotides for targeted delivery into hepatocytes or nasopharyngeal cancer cells. Enlarging the dumbbell loop size from 4 to 71 nucleotides for conjugation did not impair gene expression. GalNAc3 and aptCD137-2 residues were successfully attached to the extended dumbbell loop via complementary oligonucleotides. DNA and RNA oligonucleotide-based dumbbell-GalNAc3 conjugates were taken up from the cell culture medium by hepatoblastoma-derived human tissue culture cells (HepG2) with comparable efficiency. RNA oligonucleotide-linked conjugates triggered slightly higher levels of gene expression, presumably due to the RNaseH-mediated linker cleavage, the release of the dumbbell from the GalNAc3 residue and more efficient nuclear targeting of the unconjugated dumbbell DNA. The RNaseH-triggered RNA linker cleavage was confirmed in vitro. Finally, we featured dumbbell vectors expressing liver cancer cell-specific RNA trans-splicing-based suicide RNAs with GalNAc3 residues. Dumbbells conjugated with two GalNAc3 residues triggered significant levels of cell death when added to the cell culture medium. Dumbbell vector conjugates can be explored for targeted delivery and gene therapeutic applications.

Efficient Generation of Superior Dumbbell-Shaped Nonviral DNA Delivery Vectors Using 1-2-3 Gap-Primer PCR.

Nonviral delivery vectors are highly sought after for gene therapeutic applications and genetic vaccination. Dumbbell-shaped DNA minimal vectors have important advantages as compared with plasmids and minicircle DNA. Here, we describe the rapid, cheap, and efficient production of superior dumbbell vectors at high purity using a process termed 1-2-3 gap-primer PCR. This process represents a 1-tube, 2-enzyme, 3 h procedure that comprises a PCR followed by a ligation. The resulting dumbbells harbor mismatches close to the loop structures, which facilitate nuclear diffusion and result in enhanced gene expression.

Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA Vectors.

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.

RNA Structure Design Improves Activity and Specificity of trans-Splicing-Triggered Cell Death in a Suicide Gene Therapy Approach.

Spliceosome-mediated RNA trans-splicing enables correction or labeling of pre-mRNA, but therapeutic applications are hampered by issues related to the activity and target specificity of trans-splicing RNA (tsRNA). We employed computational RNA structure design to improve both on-target activity and specificity of tsRNA in a herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy approach targeting alpha fetoprotein (AFP), a marker of hepatocellular carcinoma (HCC) or human papillomavirus type 16 (HPV-16) pre-mRNA. While unstructured, mismatched target binding domains significantly improved 3' exon replacement (3'ER), 5' exon replacement (5'ER) correlated with the thermodynamic stability of the tsRNA 3' end. Alternative on-target trans-splicing was found to be a prevalent event. The specificity of trans-splicing with the intended target splice site was improved 10-fold by designing tsRNA that harbors secondary target binding domains shielding alternative on-target and blinding off-target splicing events. Such rationally designed suicide RNAs efficiently triggered death of HPV-16-transduced or hepatoblastoma-derived human tissue culture cells without evidence for off-target cell killing. Highest cell death activities were observed with novel dual-targeting tsRNAs programmed for trans-splicing toward AFP and a second HCC pre-mRNA biomarker. Our observations suggest trans-splicing represents a promising approach to suicide gene therapy.

Capsule-like Safe Genetic Vectors-Cell-Penetrating Core-Shell Particles Selectively Release Functional Small RNA and Entrap Its Encoding DNA.

The breakthrough of genetic therapy is set back by the lack of suitable genetic vector systems. We present the development of permeability-tunable, capsule-like, polymeric, micron-sized, core-shell particles for delivery of recombinant nucleic acids into target cells. These particles were demonstrated to effectively release rod-shaped small hairpin RNA and to selectively retain the RNA-encoding DNA template, which was designed to form a bulky tripartite structure. Thus, they can serve as delivery vectors preloaded with cargo RNA or alternatively as RNA-producing micro-bioreactors. The internalization of particles by human tissue culture cells inversely correlated with particle size and with the cell to particle ratio, although at a higher than stoichiometric excess of particles over cells, cell viability was impaired. Among primary human peripheral blood mononuclear cells, up to 50% of the monocytes displayed positive uptake of particles. Finally, these particles efficiently delivered siRNA into HEK293T cells triggering functional knockdown of the target gene lamin A/C. Particle-mediated knockdown was superior to that observed after conventional siRNA delivery via lipofection. Core-shell particles protect encapsulated nucleic acids from degradation and target cell genomes from direct contact with recombinant DNA, thus representing a promising delivery vector system that can be explored for genetic therapy and vaccination.

Expression of Herpes Simplex Virus Thymidine Kinase/Ganciclovir by RNA Trans-Splicing Induces Selective Killing of HIV-Producing Cells.

Antiviral strategies targeting hijacked cellular processes are less easily evaded by the virus than viral targets. If selective for viral functions, they can have a high therapeutic index. We used RNA trans-splicing to deliver the herpes simplex virus thymidine kinase-ganciclovir (HSV-tk/GCV) cell suicide system into HIV-producing cells. Using an extensive in silico bioinformatics and RNA structural analysis approach, ten HIV RNA trans-splicing constructs were designed targeting eight different HIV splice donor or acceptor sites and were tested in cells expressing HIV. Trans-spliced mRNAs were identified in HIV-expressing cells using qRT-PCR with successful detection of fusion RNA transcripts between HIV RNA and the HSV-tk RNA transcripts from six of ten candidate RNA trans-splicing constructs. Conventional PCR and Sanger sequencing confirmed RNA trans-splicing junctions. Measuring cell viability in the presence or absence of GCV expression of HSV-tk by RNA trans-splicing led to selective killing of HIV-producing cells using either 3' exon replacement or 5' exon replacement in the presence of GCV. Five constructs targeting four HIV splice donor and acceptor sites, D4, A5, A7, and A8, involved in regulating the generation of multiple HIV RNA transcripts proved to be effective for trans-splicing mediated selective killing of HIV-infected cells, within which individual constructs targeting D4 and A8 were the most efficient.

Engineering and Design of Polymeric Shells: Inwards Interweaving Polymers as Multilayer Nanofilm, Immobilization Matrix, or Chromatography Resins.

Hydrogels with complex internal structures are required for advanced drug delivery systems and tissue engineering or used as inks for 3D printing. However, hydrogels lack the tunability and diversity of polymeric shells and require complicated postsynthesis steps to alter its structure or properties. We report on the first integrated approach to assemble and design polymeric shells to take on various complex structures and functions such as multilayer nanofilms, multidensity immobilization matrix, or multiadhesive chromatography resins via the tuning of four assembly parameters: (a) poly(allylamine) (PA) concentration, (b) number of poly(allylamine)/poly(styrenesulfonic acid) (PA/PSSA) incubations, (c) poly(allylamine) (PA) to poly(ethylene glycol) (PEG) grafting ratio, and (d) % H2O present during assembly. Our approach combines the complex 3D structures of hydrogels with the versatility of self-assembled polymeric layers. Polymeric shells produced from our method have a highly uniform material distribution and well-defined shell boundaries. Shell thickness, density, and adhesive properties are easily tunable. By virtue of such unique material features, we demonstrate that polymeric shells can be designed to expand beyond its conventional function as thin films and serve as immobilization matrix, chromatography resins, or even reaction compartments. This technique could also uncover interesting perspectives in the development of novel multimaterials for 3D printing to synthesize scaffolds at a higher order of complexity.

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.

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.

Homologous SV40 RNA trans-splicing: Special case or prime example of viral RNA trans-splicing?

To date the Simian Virus 40 (SV40) is the only proven example of a virus that recruits the mechanism of RNA trans-splicing to diversify its sequences and gene products. Thereby, two identical viral transcripts are efficiently joined by homologous trans-splicing triggering the formation of a highly transforming 100 kDa super T antigen. Sequences of other viruses including HIV-1 and the human adenovirus type 5 were reported to be involved in heterologous trans-splicing towards cellular or viral sequences but the meaning of these events remains unclear. We computationally and experimentally investigated molecular features associated with viral RNA trans-splicing and identified a common pattern: Viral RNA trans-splicing occurs between strong cryptic or regular viral splice sites and strong regular or cryptic splice sites of the trans-splice partner sequences. The majority of these splice sites are supported by exonic splice enhancers. Splice sites that could compete with the trans-splicing sites for cis-splice reactions are weaker or inexistent. Finally, all but one of the trans-splice reactions seem to be facilitated by one or more complementary binding domains of 11 to 16 nucleotides in length which, however occur with a statistical probability close to one for the given length of the involved sequences. The chimeric RNAs generated via heterologous viral RNA trans-splicing either did not lead to fusion proteins or led to proteins of unknown function. Our data suggest that distinct viral RNAs are highly susceptible to trans-splicing and that heterologous viral trans-splicing, unlike homologous SV40 trans-splicing, represents a chance event.

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.

Homologous SV40 RNA trans-splicing: a new mechanism for diversification of viral sequences and phenotypes.

Simian Virus 40 (SV40) is a polyomavirus found in both monkeys and humans, which causes cancer in some animal models. In humans, SV40 has been reported to be associated with cancers but causality has not been proven yet. The transforming activity of SV40 is mainly due to its 94-kD large T antigen, which binds to the retinoblastoma (pRb) and p53 tumor suppressor proteins, and thereby perturbs their functions. Here we describe a 100 kD super T antigen harboring a duplication of the pRB binding domain that was associated with unusual high cell transformation activity and that was generated by a novel mechanism involving homologous RNA trans-splicing of SV40 early transcripts in transformed rodent cells. Enhanced trans-splice activity was observed in clones carrying a single point mutation in the large T antigen 5' donor splice site (ss). This mutation impaired cis-splicing in favor of an alternative trans-splice reaction via a cryptic 5'ss within a second cis-spliced SV40 pre-mRNA molecule and enabled detectable gene expression. Next to the cryptic 5'ss we identified additional trans-splice helper functions, including putative dimerization domains and a splice enhancer sequence. Our findings suggest RNA trans-splicing as a SV40-intrinsic mechanism that supports the diversification of viral RNA and phenotypes.

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.

siRNA stabilization prolongs gene knockdown in primary T lymphocytes.

RNA interference (RNAi)-mediated knockdown of target gene expression represents a powerful approach for functional genomics and therapeutic applications. However, for T lymphocytes, central regulators of immunity and immunopathologies, the application of RNAi has been limited due to the lack of efficient small interfering RNA (siRNA) delivery protocols, and an inherent inefficiency of the RNAi machinery itself. Here, we use nucleofection, an optimized electroporation approach, to deliver siRNA into primary T lymphocytes with high efficiency and negligible impairment of cell function. We identify siRNA stability within the cells as the critical parameter for efficient RNAi in primary T cells. While generally short-lived and immediately lost upon T-cell activation when conventional siRNA is used, target gene knockdown becomes insensitive to cell activation and can persist for up to 2 wk in non-dividing cells with siRNA stabilized by chemical modifications. Targeting CD4 and the transcription factor GATA-3, we show that the use of stabilized siRNA is imperative for functional gene analysis during T lymphocyte activation and differentiation in vitro as well as in vivo.

In silico selection of active siRNA.

RNA interference (RNAi) mediated by short interfering RNA (siRNA) represents a powerful reverse genetics tool, and siRNAs are attracting increasing interest as potential therapeutics. Progress in the design of functional siRNAs has significantly contributed to our understanding of cellular RNA silencing pathways and vice versa. Parameters related to RNA sequence and structure have a strong impact on various steps along the silencing pathway and build the backbone of many siRNA design tools. Recent work has demonstrated that there is more to siRNA design than enhancement of gene silencing activity. Current efforts aim at avoidance of off-target effects, the understanding of siRNA-triggered immunostimulation, and evasion of interference with cellular regulatory RNA. Molecular features determining the biological functions of siRNA and their meaning for computational (in silico) selection are the focus of this review.

RNA Silencing in the struggle against disease.

Numerous acquired and hereditary diseases are caused by aberrant cellular or microbial gene expression. As a result of sequencing of the human genome and the genomes of various human pathogens, researchers have gained access to a large number of genes with residual functions. For functional validation of unknown genes, their functions can be specifically inhibited by antisense nucleic acids or small interfering RNAs (siRNAs) and the consequences of the functional loss, that is, the resulting phenotypes, can be analyzed. While antisense nucleic acids block the translation stoichiometrically by docking on the mRNA, siRNAs induce a highly effective cellular mechanism that causes catalytic destruction of several mRNA molecules by a single siRNA molecule. This mechanism, called RNA interference (RNAi), is only intrinsic to eukaryotic cells. Consequently, only eukaryotic target validation is pushed by RNAi whereas time-consuming conventional knockout techniques or the less efficient antisense strategies have to be applied for prokaryotic target validation. We succeeded in triggering gene silencing by siRNA in prokaryotic cells. This opens promising perspectives regarding validation of prokaryotic gene functions.

Selecting effective siRNAs based on guide RNA structure.

In RNA interference, guide RNAs direct RNA-induced silencing complexes to mRNA targets, mediating cleavage and ultimately leading to gene silencing. We have observed that unstructured guide strands, which either completely lack complementary bases or in which internal base pairing is thermodynamically unlikely, confer strongest silencing, whereas structures with base-paired ends are inactive. Thus, the structure of the guide strand represents a major determinant of small interfering RNA activity. Here we describe a detailed computational protocol for identification of unstructured guide strands for a given mRNA target sequence. Sequentially, all guide sequences with target complementarity are simulated, their corresponding structures are folded and unstructured guide strands are selected and rated according to thermodynamic parameters. Although this procedure is new and remains to be validated by the community, it allows reliable identification of highly active siRNAs that can be used for functional target validation or drug development.