Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration

During the last years the potential role of in vitro transcribed (IVT) mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE). A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000) in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages of IVT mRNA as compared to pDNA as transfection agent

Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration

During the last years the potential role of in vitro transcribed (IVT) mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE). A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000) in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages of IVT mRNA as compared to pDNA as transfection agent

"Ich glaube Angst ist "not to belong""

Die vorliegende Arbeit befasst sich mit vergessenen österreichischen Kinder- und Jugendbuchautoren des britischen Exils und ihren Werken, die einerseits wieder in Erinnerung gerufen werden sollen und andererseits auf ihre exiltypische Thematologie hin analysiert werden. Im ersten Teil der Arbeit wird ein einführender Überblick über den Begriff der Exilliteratur, die österreichischen ExilautorInnen, die Publikationsumstände in Großbritannien, Kindheit im Exil und exiltypische Erzählformen gegeben. Anschließend werden im textanalytischen zweiten Teil die bedeutendsten und produktivsten Vertreter der österreichischen Kinder- und Jugendliteratur im britischen Exil - Anna Maria Jokl, Friedrich Feld (alias Fritz Rosenfeld), Hermynia Zur Mühlen, Ernst Gombrich und Marie Neurath - zunächst biographisch und anhand einiger Werke und deren Entstehungsumstände vorgestellt. Die Texte selbst werden auf die exiltypische Themen Fremdheit, Familie und Armut hin untersucht, um schließlich wiederkehrende für Exilanten identitätsstiftende Inhalte zu identifizieren. Die zugrundeliegende These, es müsse durchgängige Erzählmuster und thematische Schemata geben, konnte im Laufe der Untersuchung nicht belegt werden. Vielmehr stellte sich heraus, dass sich in den sehr unterschiedlichen narrativen Texten nur sehr geringfügige Parallelen finden lassen. Die Schlussfolgerung aus diesen Ergebnissen lässt die Mutmaßung zu, dass die verworrenen Biografien der AutorInnen und die ohnehin schwierigen Lebensumstände es nicht zuließen, sich einerseits zu einer eindeutigeren literarischen Gruppierung innerhalb der Exilliteraten zusammenzufinden und es andererseits auch nicht möglich war, sich so schnell in einem fremden Literaturbetrieb neu zu orientieren. Somit blieben die meisten AutorInnen ihrer Linie treu, die sie auch schon vor dem Exil pflegten. Trends in der Auswahl des Genres, der Gattung oder der Themen konnten nicht belegt werden

Genetic Determinants and Epidemiology of Cystic Fibrosis–Related Diabetes: Results from a British cohort of children and adults

OBJECTIVE—Longer survival of patients with cystic fibrosis has increased the occurrence of cystic fibrosis–related diabetes (CFRD). In this study we documented the incidence of CFRD and evaluated the association between mutations responsible for cystic fibrosis and incident CFRD, while identifying potential risk factors

Fracture properties of CrN hard coatings: Influence of the microstructure, alloying elements, and coating architecture

Transition metal nitrides are well known and applied as protective coating materials based on their unique refractory characteristics, such as high hardness or Young’s modulus. However, for long-term applications, the fracture toughness KIC is an essential factor as the integrity of the coating-substrate interface is impaired by cracking and subsequent environmental attacks. Please click Download on the upper right corner to see the full abstract

Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei

We investigated in different human cell types nuclear positioning and transcriptional regulation of the functionally unrelated genes GASZ, CFTR, and CORTBP2, mapping to adjacent loci on human chromosome 7q31. When inactive, GASZ, CFTR, and CORTBP2 preferentially associated with the nuclear periphery and with perinuclear heterochromatin, whereas in their actively transcribed states the gene loci preferentially associated with euchromatin in the nuclear interior. Adjacent genes associated simultaneously with these distinct chromatin fractions localizing at different nuclear regions, in accordance with their individual transcriptional regulation. Although the nuclear localization of CFTR changed after altering its transcription levels, the transcriptional status of CFTR was not changed by driving this gene into a different nuclear environment. This implied that the transcriptional activity affected the nuclear positioning, and not vice versa. Together, the results show that small chromosomal subregions can display highly flexible nuclear organizations that are regulated at the level of individual genes in a transcription-dependent manner

Cystic fibrosis and renal disease: a case report

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Cell type differences in activity of the Streptomyces bacteriophage ϕC31 integrase

Genomic integration by the Streptomyces bacteriophage ϕC31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the ϕC31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34+ haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of ϕC31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in ϕC31 integrase transfected A549 lung than Jurkat T cells. When the ϕC31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the ϕC31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of ϕC31 integrase activity

Characterization of Ku702–NLS as Bipartite Nuclear Localization Sequence for Non-Viral Gene Delivery

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku702-NLS), a nuclear transport active mutant of Ku702-NLS (s1Ku702-NLS) and a nuclear transport deficient mutant of Ku702-NLS (s2Ku702). We examined the transfection efficiency of binary Ku702-NLS/DNA and ternary Ku702-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku702-NLS and s1Ku702-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer