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  • 1
    Keywords: IN-VITRO ; LONG-TERM ; SMALL INTERFERING RNAS ; HEPATITIS-C VIRUS ; ARGONAUTE PROTEINS ; GUIDE STRAND SELECTION ; SMALL HAIRPIN RNAS ; ADENOASSOCIATED VIRUSES ; EFFICIENT INHIBITION ; MICRORNA BIOGENESIS
    Abstract: Exogenous RNAi triggers such as shRNAs ideally exert their activities exclusively via the antisense strand that binds and silences designated target mRNAs. However, in principle, the sense strand also possesses silencing capacity that may contribute to adverse RNAi side effects including off-target gene regulation. Here, we address this concern with a novel strategy that reduces sense strand activity of vector-encoded shRNAs via codelivery of inhibitory tough decoy (TuD) RNAs. Using various shRNAs for proof of concept, we validate that coexpression of TuDs can sequester and inactivate shRNA sense strands in human cells selectively without affecting desired antisense activities from the same shRNAs. Moreover, we show how coexpressed TuDs can alleviate shRNA-mediated perturbation of global gene expression by specifically de-repressing off-target transcripts carrying seed matches to the shRNA sense strand. Our combination of shRNA and TuD in a single bicistronic gene transfer vector derived from Adeno-associated virus (AAV) enables a wide range of applications, including gene therapies. To this end, we engineered our constructs in a modular fashion and identified simple hairpin design rules permitting adaptation to preexisting or new shRNAs. Finally, we demonstrate the power of our vectors for combinatorial RNAi strategies by showing robust suppression of hepatitis C virus (HCV) with an AAV expressing a bifunctional TuD against an anti-HCV shRNA sense strand and an HCV-related cellular miRNA. The data and tools reported here represent an important step toward the next generation of RNAi triggers with increased specificity and thus ultimately safety in humans.
    Type of Publication: Journal article published
    PubMed ID: 26170322
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  • 2
    Abstract: Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) scaffold, into an engineered genomic miRNA locus. For proof-of-concept, we used TALE or CRISPR/Cas9 nucleases to site-specifically integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr locus in hepatoma cells, with the aim to obtain cellular clones that are genetically protected against HCV infection. Using reporter assays, Northern blotting and qRT-PCR, we confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality in selected cellular progeny. Moreover, we employed a comprehensive battery of PCR, cDNA/miRNA profiling and whole genome sequencing analyses to validate targeted integration of a single shmiRNA molecule at the expected position, and to rule out deleterious effects on the genomes or transcriptomes of the engineered cells. Importantly, a subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNAi expression technologies benefits numerous applications, from miRNA, genome and transgenesis research, to human gene therapy.
    Type of Publication: Journal article published
    PubMed ID: 27614072
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  • 3
    Keywords: CELLS ; liver ; MUTATIONS ; IMMUNITY ; GENE-THERAPY ; VIRUS VECTORS ; ENDONUCLEASE ; GUIDE ; CAS SYSTEMS ; CRISPR
    Abstract: Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans.
    Type of Publication: Journal article published
    PubMed ID: 25186301
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  • 4
    Abstract: BACKGROUND: The CRISPR/Cas9 genome editing system has greatly facilitated and expanded our capacity to engineer mammalian genomes, including targeted gene knock-outs. However, the phenotyping of the knock-out effect requires a high DNA editing efficiency. RESULTS: Here, we report a user-friendly strategy based on the extrinsic apoptosis pathway that allows enrichment of a polyclonal gene-edited cell population, by selecting Cas9-transfected cells that co-express dominant-negative mutants of death receptors. The extrinsic apoptosis pathway can be triggered in many mammalian cell types, and ligands are easy to produce, do not require purification and kill much faster than the state-of-the-art selection drug puromycin. Stringent assessment of our advanced selection strategy via Sanger sequencing, T7 endonuclease I (T7E1) assay and direct phenotyping confirmed a strong and rapid enrichment of Cas9-expressing cell populations, in some cases reaching up to 100 % within one hour. Notably, the efficiency of target DNA cleavage in these enriched cells reached high levels that exceeded the reliable range of the T7E1 assay, a conclusion that can be generalized for editing efficiencies above 30 %. Moreover, our data emphasize that the insertion and deletion pattern induced by a specific gRNA is reproducible across different cell lines. CONCLUSIONS: The workflow and the findings reported here should streamline a wide array of future low- or high-throughput gene knock-out screens, and should largely improve data interpretation from CRISPR experiments.
    Type of Publication: Journal article published
    PubMed ID: 26883910
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  • 5
    Abstract: Hepatitis B virus (HBV) is a promising target for therapies based on RNA interference (RNAi) since it replicates via RNA transcripts that are vulnerable to RNAi silencing. Clinical translation of RNAi technology, however, requires improvements in potency, specificity and safety. To this end, we systematically compared different strategies to express anti-HBV short hairpin RNA (shRNA) in a pre-clinical immunocompetent hepatitis B mouse model. Using recombinant Adeno-associated virus (AAV) 8 vectors for delivery, we either (i) embedded the shRNA in an artificial mi(cro)RNA under a liver-specific promoter; (ii) co-expressed Argonaute-2, a rate-limiting cellular factor whose saturation with excess RNAi triggers can be toxic; or (iii) co-delivered a decoy ("TuD") directed against the shRNA sense strand to curb off-target gene regulation. Remarkably, all three strategies minimised adverse side effects as compared to a conventional shRNA vector that caused weight loss, liver damage and dysregulation of 〉 100 hepatic genes. Importantly, the novel AAV8 vector co-expressing anti-HBV shRNA and TuD outperformed all other strategies regarding efficiency and persistence of HBV knock-down, thus showing substantial promise for clinical translation.
    Type of Publication: Journal article published
    PubMed ID: 27473329
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  • 6
  • 7
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    German Medical Science GMS Publishing House; Düsseldorf
    In:  87. Jahresversammlung der Deutschen Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und Hals-Chirurgie; 20160504-20160507; Düsseldorf; DOC16hnod095 /20160330/
    Publication Date: 2016-03-31
    Keywords: ddc: 610
    Language: German
    Type: conferenceObject
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  • 8
    ISSN: 0392-6737
    Keywords: Electron states in low-dimensional structures (including quantum wells, superlattices, layer structures, and intercalation compounds) ; Localized single-particle electronic states (including impurities) ; III–V compounds and systems ; Photoluminescence ; Time-resolved optical spectroscopies and other ultrafast optical measurements in condensed matter ; Conference proceedings
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Summary We have performed time-integrated and time-resolved photoluminescence experiments on high-quality stressor-induced InGaAs/GaAs quantum dots. The optical spectra of these dot structures exhibit large quantization effects together with remarkably low inhomogeneous broadenings of the respective excitonic transitions. Thus a detailed investigation of the relaxation and recombination dynamics within the distinct electronic dot states becomes feasible. We find that the initial relaxation of optically generated carriers down to the lowest dot states is very efficient. This fast thermalization is ascribed to Coulomb scattering between carriers confined in dot states and carriers located in the higher-energetic quantum well states.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 0021-8383
    Keywords: Chemistry ; Organic Chemistry
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Description / Table of Contents: Studies on Sulphochlorination of Paraffins. I. Kinetic Studies on the Monosulphochlorination of the n-Paraffins C6—C16The monosulphochlorides in the reaction mixtures of the sulphochlorination of n-paraffins may be transformed into the gaschromatographic separable sulphonic acid dimethyl amides by reaction with dimethylamine in ether.So the analysis of the isomers in the mixtures from sulphochlorination of n-paraffins C5—C16 becomes possible. The results together with the relative rates of sulphochlorination of n-paraffins C6—C16 (determined by competitive reaction) allow the calculation of sulphochlorination rates of the different C—H-bonds in the n-paraffins C6—C16 relative to one primary C—H-bond in n-octane. For the n-paraffins C6—C8 the relative rates of sulphochlorination of different C—H-bonds agree with the corresponding relative rates of chlorination.
    Notes: Die in Sulfochlorierungsgemischen von n-Paraffinen enthaltenen isomeren Monosulfochloride lassen sich durch Umsetzen mit Dimethylamin/Äther in die gaschromatographisch trennbaren Sulfonsäuredimethylamide überführen. So ist die Isomerenanalyse der Sulfochlorierungsgemische aus den n-Paraffinen C5—C16 möglich. Die Ergebnisse sowie die durch Konkurrenzreaktion ermittelten relativen Sulfochlorierungsgeschwindigkeiten der n-Paraffine C6—C16 gestatten die Berechnung der auf eine primäre C—H-Bindung des n-Octans bezogenen relativen Sulfochlorierungsgeschwindigkeiten der verschiedenen C—H-Bindungen in den Paraffinen C6—C16. Bei den Paraffinen C6—C8 stimmen die relativen Reaktionsgeschwindigkeiten der verschiedenen C—H-Bindungen bei der Sulfochlorierung und bei der Chlorierung innerhalb der Fehlergrenzen überein.
    Additional Material: 7 Tab.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1089-7674
    Source: AIP Digital Archive
    Topics: Physics
    Notes: A fluid model of surface wave produced discharges is presented for diffusion controlled regimes taking into account simultaneously nonlinear contributions from stepwise ionization and volume recombination. The saturation of metastable densities with growing electron density reduces the effect of step ionization and allows recombination to become effective toward high electron densities. The charged particle continuity and energy balance equations linking electron density and electric field intensity yield the plasma permittivity under conditions of strong ionization nonlinearity. This permittivity is valid for high frequency discharges in general. In the second part of the modeling this nonlinear permittivity is introduced into the electrodynamical relations for discharges maintained by the field of traveling surface waves, and subsequently the self-consistent behavior of plasma and wave characteristics along the discharge length is calculated. Both the main part of the discharge column produced by Joule heating in the plasma volume and the end section of the discharge, where the mechanism of resonance absorption of the wave power close to the tube wall can sustain the discharge, are considered. © 1996 American Institute of Physics.
    Type of Medium: Electronic Resource
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