Enhancing non‐viral cell transfection through lysosomal escape mediated by listeriolysin O

Im Gegensatz zu Strategien basierend auf die Einführung von transgenenen Zellen, die Wachstumsfaktoren exprimieren, oder die direkte Administration von rekombinanten Wachstumsfaktoren in vivo, bietet die Gentherapie (Einführung von exogener DNA kodierend für einen Wachstumsfaktor) eine einfach anzuwendende, kostengünstige Alternative, verbunden mit erhöhter Bioaktivität der exprimierten Wachstumsfaktoren (durch wirtszell-spezifischer post-translationaler Modifikation und korrekter Faltung der lokal durch die Zielzellen produzierten Wachstumsfaktoren). Jedoch ist der Einsatz von viralen Vektoren, die eine effiziente Einschleusung von Fremd-DNA in Zielzellen in vivo garantieren, wegen der Auslösung einer Immunantwort und das Integrieren der Fremd-DNA in das Genom der Zielzelle, ungeeignet. Nicht-virale Vektoren hingegen bieten eine hohe Sicherheit und erzeugen eine geringfügige Immunantwort, sind jedoch in ihrer Fähigkeit Zielzellen in vivo zu transfizieren, beschränkt. Die physische und chemische Barriere der Zellmembran, der lysosomale Abbau und die geringe Effizienz der Diffusion der eingeführten DNA in den Zellkern limitiert die Wirksamkeit der Transfektion erheblich. Diese Arbeit war ausgerichtet, den lysosomalen Abbau eingeführter Makromoleküle wie DNA zu verhindern um die Transfektionseffizienz zu erhöhen. Um den lysosomalen Abbau zu vermeiden und dadurch die Wahrscheinlichkeit der Einführung therapeutischer DNA in das Zytosol bzw. den Zellkern zu erhöhen, wurde versucht, mithilfe eines bakteriellen Proteins (Listeriolysin O) die Lysosomen während der endozytischen Aufnahme der DNA zu zerstören. Durch die pH-abhängige Aktivierung der Hämolysins in der lysosomalen Umgebung werden die Lysosomen zerstört und die endozytotisch aufgenommene therapeutische DNA ins Zytosol freigesetzt. Listeriolysin O wurde in E. coli exprimiert und anschließend biochemisch aufgereinigt, um dessen Einfluss auf die DNA-Transfektion in eukaryotischen Zellen zu ermitteln. Bei Anwesenheit von Listeriolysin O zeigte sich eine erhöhte Präsenz von aufgenommenen Molekülen (DNA, Farbstoff) im Zytosol der Zielzellen.In contrast to strategies based on the introduction of transgenic cells expressing growth factors (ex vivo therapy), or the direct administration of recombinant growth factors into target systems, in vivo gene therapy approaches (introduction of therapeutic plasmids encoded for growth factors) provide a promising alternative associated with lower manufacturing costs, higher safety and increased bioactivity of the produced proteins (due to host-specific post-translational modifications and correct folding of the locally produced growth factors). Utilizing viral particles for high transfection efficiencies (high efficiency for DNA introduction into target cells in vivo) is unsuitable due to the high immunogenicity and the nature of some viral vectors to manipulate the host genome. On the other hand, non-viral gene delivery methods provide high safety and low immune response, but are limited in their transfection efficiency. Main reasons for impaired transfection capacity are confined cellular uptake of the exogenous DNA, the lysosomal degradation after uptake and the low efficiency of the introduced plasmid DNA to diffuse into the cell nucleus. This study was focused on the lysosomal degradation which highly limits the transfection efficiency. In order to avoid the lysosomal degradation of introduced therapeutic DNA and therefore to significantly increase the probability of the engulfed DNA to overcome the lysosomal barrier, the influence of the hemolytic protein listeriolysin O, derived from the intracellular bacteria Listeria monocytogenes, was tested in gene delivery approaches. During the life cycle of Listeria monocytogenes, the hemolytic protein is secreted after the engulfment of the bacteria in order to disrupt the lysosomes and to ensure the bacterial entry into the cytosol of target cells. The main advantage of listeriolysin O is its hemolytic activity to disrupt eukaryotic membranes restricted only under acidic conditions (below pH = 6) as given in the lysosomes. Due to this pH dependent activity of listeriolysin O, and the subsequent deactivation by aggregation after exposition to physiological pH conditions (pH = 7), the subsequent disruption of the host cell membrane is avoided preventing the host cell from lysis. Based on this, listeriolysin O is a promising auxiliary protein for the enhancement of the transfection efficiency. The hlyA gene, encoded for Listeriolysin O and lacking its secretion signal, was C-terminally linked to a polyhistidine tag and cloned into a bacterial expression vector following expression in E. coli. Subsequent optimization of the protein purification was performed to attain high yields of pure and bioactive LLO, extensively exceeding presently published results concerning total yield of LLO and effort of the purification method. The influence of the purified LLO in cell transfection approaches was tested in vitro. The DNA was prelabeled with the fluorescence dye and complexed with the polycationic DNA carrier poly-L-lysine (PLL). Particles were transferred to the cells and the uptake efficiency of the particles was observed in the presence and absence of LLO after 15minutes under fluorescence excitation to confirm the subsequent access of the DNA dye into the cell nucleus after lysosomal disruption by LLO. By comparing the uptake of the fluorescence labeled DNA microparticles, a remarkable increase of the uptake was observed within the first 15 minutes under the fluorescence microscope when LLO and the DNA microparticles were simultaneously transferred to the cells. In contrast, transfection of cells in the absence of LLO showed an entrapment of the microparticles inside the lysosomes within the same time period

Improved osteogenic vector for non-viral gene therapy

Therapeutic compensation of deficient bone regeneration is a challenging task and a topic of on-going search for novel treatment strategies. One promising approach for improvement involves non-viral gene delivery using the bone morphogenetic protein-2 (BMP-2) gene to provide transient, local and sustained expression of the growth factor. However, since efficiency of non-viral gene delivery is low, this study focused on the improvement of a BMP-2 gene expression system, aiming for compensation of poor transfection efficiency. First, the native BMP-2 gene sequence was modified by codon optimisation and altered by inserting a highly truncated artificial intron (96 bp). Transfection of multiple cell lines and rat adipose-derived mesenchymal stem cells with plasmids harbouring the improved BMP-2 sequence led to a several fold increased expression rate and subsequent osteogenic differentiation. Additionally, comparing expression kinetics of elongation factor 1 alpha (EF1α) promoter with a state of the art CMV promoter revealed significantly higher BMP-2 expression when under the influence of the EF1α promoter. Results obtained by quantification of bone markers as well as osteogenic assays showed reduced sensitivity to promoter silencing effects of the EF1α promoter in rat adipose-derived mesenchymal stem cells. Finally, screening of several protein secretion signals using either luciferase or BMP-2 as reporter protein revealed no superior candidates for potential replacement of the native BMP-2 secretion signal. Taken together, by enhancing the exogenous BMP-2 expression system, low transfection efficiencies in therapeutic applications can be compensated, making safe non-viral systems even more suitable for tissue regeneration approaches

A luciferase-based quick potency assay to predict chondrogenic differentiation.

Chondrogenic differentiation of adipose derived stem cells (ASC) is challenging but highly promising for cartilage repair. Large donor variability of chondrogenic differentiation potential raises the risk for transplantation of cells with reduced efficacy and a low chondrogenic potential. Therefore quick potency assays are required in order to control the potency of the isolated cells before cell transplantation. Current in vitro methods to analyze the differentiation potential are time consuming and thus, a novel enhancer and tissue-specific promoter combination was employed for the detection of chondrogenic differentiation of ASC in a novel quick potency bioassay. Human primary ASC were co-transfected with the Metridia luciferase based collagen type II reporter gene pCMVE_ACDCII-MetLuc together with a Renilla control plasmid and analyzed for their chondrogenic potential. On day 3 after chondrogenic induction, the luciferase activity was induced in all tested donors under three dimensional (3D) culture conditions and in a second approach also under 2D culture conditions. With our newly developed quick potency bioassay we can determine chondrogenic potential already after 3 days of chondrogenic induction and under 2D culture conditions. This will enhance the efficiency of testing cell functionality, which should allow in the future to predict the suitability of cells derived from individual patients for cell therapies, in a very short time and at low costs

Spatiotemporal Differences in Gene Expression Between Motor and Sensory Autografts and Their Effect on Femoral Nerve Regeneration in the Rat

To improve the outcome after autologous nerve grafting in the clinic, it is important to understand the limiting variables such as distinct phenotypes of motor and sensory Schwann cells. This study investigated the properties of phenotypically different autografts in a 6 mm femoral nerve defect model in the rat, where the respective femoral branches distally of the inguinal bifurcation served as homotopic, or heterotopic autografts. Axonal regeneration and target reinnervation was analyzed by gait analysis, electrophysiology, and wet muscle mass analysis. We evaluated regeneration-associated gene expression between 5 days and 10 weeks after repair, in the autografts as well as the proximal, and distal segments of the femoral nerve using qRT-PCR. Furthermore we investigated expression patterns of phenotypically pure ventral and dorsal roots. We identified highly significant differences in gene expression of a variety of regeneration-associated genes along the central – peripheral axis in healthy femoral nerves. Phenotypically mismatched grafting resulted in altered spatiotemporal expression of neurotrophic factor BDNF, GDNF receptor GFRα1, cell adhesion molecules Cadm3, Cadm4, L1CAM, and proliferation associated Ki67. Although significantly higher quadriceps muscle mass following homotopic nerve grafting was measured, we did not observe differences in gait analysis, and electrophysiological parameters between treatment paradigms. Our study provides evidence for phenotypic commitment of autologous nerve grafts after injury and gives a conclusive overview of temporal expression of several important regeneration-associated genes after repair with sensory or motor graft

Mitochondria-Targeted Antioxidants SkQ1 and MitoTEMPO Failed to Exert a Long-Term Beneficial Effect in Murine Polymicrobial Sepsis

Mitochondrial-derived reactive oxygen species have been deemed an important contributor in sepsis pathogenesis. We investigated whether two mitochondria-targeted antioxidants (mtAOX; SkQ1 and MitoTEMPO) improved long-term outcome, lessened inflammation, and improved organ homeostasis in polymicrobial murine sepsis. 3-month-old female CD-1 mice (n=90) underwent cecal ligation and puncture (CLP) and received SkQ1 (5 nmol/kg), MitoTEMPO (50 nmol/kg), or vehicle 5 times post-CLP. Separately, 52 SkQ1-treated CLP mice were sacrificed at 24 h and 48 h for additional endpoints. Neither MitoTEMPO nor SkQ1 exerted any protracted survival benefit. Conversely, SkQ1 exacerbated 28-day mortality by 29%. CLP induced release of 10 circulating cytokines, increased urea, ALT, and LDH, and decreased glucose but irrespectively of treatment. Similar occurred for CLP-induced lymphopenia/neutrophilia and the NO blood release. At 48 h post-CLP, dying mice had approximately 100-fold more CFUs in the spleen than survivors, but this was not SkQ1 related. At 48 h, macrophage and granulocyte counts increased in the peritoneal lavage but irrespectively of SkQ1. Similarly, hepatic mitophagy was not altered by SkQ1 at 24 h. The absence of survival benefit of mtAOX may be due to the extended treatment and/or a relatively moderate-risk-of-death CLP cohort. Long-term effect of mtAOX in abdominal sepsis appears different to sepsis/inflammation models arising from other body compartments

Enhancement of orthopaedic gene therapy

Eine der Hauptkomponenten der Gewebezüchtung beinhaltet die Anwendung von gewebespezifischen Wachstumsfaktoren entweder auf Proteinebene oder kodiert auf einem theraoeutischen DNA-Plasmid (Gentherapie). Die Anwendung von therapeutischen Plasmiden kodierend für Wachstumsfaktoren bietet ein vielverpsrechendes Mittel verbunden mit niedrigen Herstellungskosten, höherer Sicherheit und erhöhte Bioaktivität der produzierten Proteine wegen wirtsspezifischer post-translationaler Modifikationen und korrekter Faltung der lokal produzierten Wachstumsfaktoren. Um exogene DNA in Zielzellen (sowohl in vitro als auch in vivo) einzubringen, können mehrere virale oder nicht-virale Gentransfer-Methoden angewendet werden. Jedoch sind nicht-virale Methoden in ihrer Effektivität beschränkt. Trotzdem punkten nicht-virale Transfersysteme durch niedrige Produktionskosten, leichter Anwendbarkeit und Sicherheit. Deshalb sind viele Versuche laufend, die auf spezifische Modifikationen der Transfersysteme oder durch das Zufügen von Helfersubstanzen (z.B Peptide), um die Wirksamkeit des nicht-viralen Gentransfers zu erhöhen, als viralen Gentransfer sicherer zu machen. Aus diesen Gründen wurden in dieser Arbeit Methoden vorgestellt um einen schwachen Gentransfer, oder die Effizienz der Genexpression, zu verbessern um die schwache Wirksamkeit zu kompensieren. Erstens wurde die hämolytische Aktivität verschiedener, auf Virulenzfaktoren basierender Peptide miteinander verglichen. sogenannte endosomolytischen Peptide mediieren die Zerstörung der Lysosomenmembran während des Gentransfers und verhindern dadurch den Abbau des Gentransfersystems sowie der therapeutischen DNA. Weiters wurden Strategien entwickelt, um die Genexpression an sich zu erhöhen, um einen eventuellen schlechten Gentransfer vorzubeugen, unter anderem Modifikationen am Promotersystem der Expressionskasette (Kaskadenpromoter). Weiters wurde die Stärke von Sekretionssignalen von unterschiedlichen Protein verglichen, um eventuell schwache Signalsequenzen der eingesetzten Wachstumsfaktoren mit potenteren zu ersetzen. Dabei wurden unter anderem auch interessante Engpässe in der Proteinsekretion beobachtet und analysiert. Letztendlich wurden auch im Fall der Knochenregeneration Strategien vorgestellt, um nicht-virale Anwendungen der Gentherapie speziell in diesem Fall (osteogene Differenzierung) zu verbessern. Unter anderem der Einsatz und Verifizierung von Hybridplasmiden, konzipiert für eine transiente Überexpression eines knochenspezifischen Wachstumsfaktors bei gleichzeitiger erzwungener Unterdrückung (Herunterregulierung) von potentiellen Inhibitoren des osteogenen Signalweges.One of the key components of tissue engineering is the delivery of tissue specific growth factors either as protein level or encoded on therapeutic DNA plasmids (gene therapy). The application of therapeutic plasmids encoded for growth factors provides a promising tool associated with lower manufacturing costs, higher safety and increased bioactivity of the produced proteins due to host-specific post-translational modifications and correct folding of the locally produced growth factors. In order to introduce exogenous DNA into cells in vitro and in vivo, several strategies based on viral or non-viral gene transfer approaches can be applied. Unfortunately, non-viral gene transfer methods are limited in their efficacy. Nevertheless, non-viral vectors score concerning their manufacturing cost, easy handling and safety. Therefore, many attempts are ongoing to increase the efficacy of gene delivery using non-viral vectors by specific modifications or additional auxiliary substances, than trying to make viral systems safer. Therefore, in this work, different strategies are introduced to compensate weak gene delivery. Firstly, hemolytic activity of different viruence-factor derived peptides were compared for their potency to enhance in gene transfer systems as mediating disruption of lysosomes. Compared to other tested endosomolytic peptides, the virulence-factor Listeriolysin O showed unrivalled pH specific hemolytic activity. Secondly, strategies to enhance gene expression itself were introduced in order to compensate weak transfection efficiency. Sequence modifications of the therapeutic gene showed several fold increase in gene expression. Additionally, by constructing a cascade promoter system, bottlenecks of transfection factor depletion was overcomed, leading to further increase in expression. Thirdly, screening of potent secretion signal sequences to mediate enhanced protein secretion by signal sequence exchange was performed. Additionally, by monitoring protein secretion kinetics intersting insights into bottlenecks during protein secretion were observed. Finally, additional strategies to improve specifically osteogenic differentiation. Amongst others, the application of a hybrid vector system for overexpression of a therapeutic gene by simulatenous downregulation of inhibitory factors showed enhanced osteogenic differentiation of target cells compared to conventional vectors

Modification of plasmid elements for prolonged gene expression in mammalian cell systems

Die Produktion rekombinanter Proteine in Säugerzellsystemen ist zu einer Routine geworden. Dies liegt vor allem in ihrem vorteilhaften Verhältnis von Arbeitszeit zu Ausbeute begründet. Die Vorteile von exprimierten Proteinen, die der nativen Form sehr ähnlich sind, zeigen oft eine höhere Bioaktivität und sind daher wichtig für die Herstellung biofunktioneller Materialien für Biopharmazeutika oder klinische Anwendungen. Die Produktion rekombinanter Proteine in Säugerzellsystemen durch transiente Genexpression wird aufgrund des schnellen Herstellungsprozesses immer beliebter. Das herkömmliche Verfahren, das auf stabiler Genexpression basiert, ist zeitaufwändig und für die Auswahl und das Screening des korrekten stabilen Zellklons sind viele Ressourcen erforderlich. Da die meisten hergestellten Proteine hauptsächlich für Forschungsanwendungen verwendet werden, kann es riskant sein, in Zeit und Ressourcen zu investieren, ohne zu wissen ob im Nachhinein das Protein für klinische Ansätze zugelassen wird. Aufgrund dieses Umstandes ist die transiente Genexpression ein wesentliches Werkzeug, um den Bedarf an Proteinen und Peptiden für Forschungsanwendungen zu decken. In dieser Arbeit wurde eine modifizierte Version des Kanamycin-Resistenzgens entwickelt und in verschiedene Plasmidsysteme implementiert. Der Kanamycinresistenz-Gensequenz fehlen CpG-Nukleotide, die sich als Hot-Spots für Methylierungsprozesse in Säugetierzielzellen verantwortlich zeigen. Diese Arbeit zeigt, dass Säugetier-Expressionsplasmide, die diese neuartige genetische Sequenz beinhalten, weniger anfällig für transkriptionelles Silencing sind, verglichen mit Plasmiden mit dem herkömmlichen Kanamycinresistenzgen. Dieser Effekt wurde durch die Expression verschiedener Reportergene, sowie eines therapeutischen Wachstumsfaktorproteins (Bone morphogenetic protein 2) unter Verwendung verschiedener Visualisierungs- und Quantifizierungsmethoden gezeigt. Plasmide, die das neu gestaltete Kanamycin-Gen beinhalten, zeigen signifikant verringerte transkriptionelle Inaktivierung, was zu einer mehrfach höheren Gesamtausbeute an exprimiertem Protein führt. Dies beruhte hauptsächlich auf dem Phänomen, dass die Transgenexpression auf Plasmiden mit dem nativen Kanamycin-Resistenzgen nach einer kurzen Zeitspanne von 2-3 Tagen ausgeschaltet wurde, verglichen mit der verlängerten Genexpression mit den neuen Plasmiden von mindestens mehreren Wochen. Die in dieser Studie erzielten Ergebnisse zeigen den Vorteil des Austauschs des herkömmlichen Antibiotikagens aufgrund ihrer stark erhöhten Effizienz und der verlängerten Expression von durch Wirtszellen produzierten therapeutischen Proteinen.Recombinant protein production in mammalian cell systems has become a routine and efficient in terms of time to yield ratio. The advantages of expressed proteins that closely resembles the native form often show higher bioactivity and are therefore important for the fabrication of biofunctional materials for biopharmaceuticals or clinical applications. Producing recombinant proteins in mammalian cell systems by transient gene expression is gaining popularity due to its advantages compared to stable gene expression. The latter, which is still the conventional approach is time consuming and a lot of resources are necessary for the selection and screening process of the correct stable cell clone. Since most candidate proteins are primarily used for research applications only, it can be risky to make investments in time and resources without assertion that the protein of interest will be approved for clinical approaches. Based on this, transient gene expression is an essential tool in order to address the need for proteins and peptides for research applications. In this work, a modified version of the kanamycin resistance gene was developed and implemented in different plasmid systems. The kanamycin resistance gene sequence is lacking CpG-nucleotides, which are known as hot-spots for methylation processes in mammalian target cells. This work demonstrates that mammalian expression plasmids harbouring this novel genetic sequence are less prone to transcriptional silencing (shut-down), when compared to plasmids harbouring the conventional kanamycin resistance gene. This effect was shown by expressing different reporter genes as well as a therapeutical growth factor protein (Bone morphogenetic protein 2) using different visualization and quantification methods. Plasmids harbouring the newly designed kanamycin gene show significantly decreased transcriptional silencing, resulting in a several fold higher overall yield of expressed protein. This was primarily based on the phenomenon that transgene expression on plasmids with the native kanamycin resistance gene were silenced after a short time period of 2-3 days, while the new plasmids showed a prolonged gene expression of minimum several weeks. The results obtained in this study strongly suggest an advantage of replacing the conventional antibiotic gene, generating plasmids with a higher efficiency and prolonged expression of host cell produced therapeutic proteins.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersWien, FH Campus Wien, Masterarb., 2019(VLID)435483

Resistance of Bacteria toward 475 nm Blue Light Exposure and the Possible Role of the SOS Response

The increase in antibiotic resistance represents a major global challenge for our health systems and calls for alternative treatment options, such as antimicrobial light-based therapies. Blue light has shown promising results regarding the inactivation of a variety of microorganisms; however, most often, antimicrobial blue light (aBL) therapy is performed using wavelengths close to the UV range. Here we investigated whether inactivation was possible using blue light with a wavelength of 475 nm. Both Gram-positive and -negative bacterial strains were treated with blue light with fluences of 7.5–45 J/cm2. Interestingly, only some bacterial strains were susceptible to 475 nm blue light, which was associated with the lack of RecA, i.e., a fully functional DNA repair mechanism. We demonstrated that the insertion of the gene recA reduced the susceptibility of otherwise responsive bacterial strains, indicating a protective mechanism conveyed by the bacterial SOS response. However, mitigating this pathway via three known RecA inhibiting molecules (ZnAc, curcumin, and Fe(III)-PcTs) did not result in an increase in bactericidal action. Nonetheless, creating synergistic effects by combining a multitarget therapy, such as aBL, with an RecA targeting treatment could be a promising strategy to overcome the dilemma of antibiotic resistance in the future

Spatiotemporal Differences in Gene Expression Between Motor and Sensory Autografts and Their Effect on Femoral Nerve Regeneration in the Rat.

To improve the outcome after autologous nerve grafting in the clinic, it is important to understand the limiting variables such as distinct phenotypes of motor and sensory Schwann cells. This study investigated the properties of phenotypically different autografts in a 6 mm femoral nerve defect model in the rat, where the respective femoral branches distally of the inguinal bifurcation served as homotopic, or heterotopic autografts. Axonal regeneration and target reinnervation was analyzed by gait analysis, electrophysiology, and wet muscle mass analysis. We evaluated regeneration-associated gene expression between 5 days and 10 weeks after repair, in the autografts as well as the proximal, and distal segments of the femoral nerve using qRT-PCR. Furthermore we investigated expression patterns of phenotypically pure ventral and dorsal roots. We identified highly significant differences in gene expression of a variety of regeneration-associated genes along the central - peripheral axis in healthy femoral nerves. Phenotypically mismatched grafting resulted in altered spatiotemporal expression of neurotrophic factor BDNF, GDNF receptor GFRα1, cell adhesion molecules Cadm3, Cadm4, L1CAM, and proliferation associated Ki67. Although significantly higher quadriceps muscle mass following homotopic nerve grafting was measured, we did not observe differences in gait analysis, and electrophysiological parameters between treatment paradigms. Our study provides evidence for phenotypic commitment of autologous nerve grafts after injury and gives a conclusive overview of temporal expression of several important regeneration-associated genes after repair with sensory or motor graft