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

Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy

Tissue regenerative gene therapy requires expression strategies that deliver therapeutic effective amounts of transgenes. As physiological expression patterns are more complex than high-level expression of a singular therapeutic gene, we aimed at constitutive or inducible co-expression of 2 transgenes simultaneously. Co-expression of human bone morphogenetic protein 2 and 7 (BMP2/7) from constitutively expressing and doxycycline inducible plasmids was evaluated in vitro in C2C12 cells with osteocalcin reporter gene assays and standard assays for osteogenic differentiation. The constitutive systems were additionally tested in an in vivo pilot for ectopic bone formation after repeated naked DNA injection to murine muscle tissue. Inductor controlled differentiation was demonstrated in vitro for inducible co-expression. Both co-expression systems, inducible and constitutive, achieved significantly better osteogenic differentiation than single factor expression. The potency of the constitutive co-expression systems was dependent on relative expression cassette topology. In vivo, ectopic bone formation was demonstrated in 6/13 animals (46 % bone formation efficacy) at days 14 and 28 in hind limb muscles as proven by in vivo μCT and histological evaluation. In vitro findings demonstrated that the devised single vector BMP2/7 co-expression strategy mediates superior osteoinduction, can be applied in an inductor controlled fashion and that its efficiency is dependent on expression cassette topology. In vivo results indicate that co-expression of BMP2/7 applied by non-viral naked DNA gene transfer effectively mediates bone formation without the application of biomaterials, cells or recombinant growth factors, offering a promising alternative to current treatment strategies with potential for clinical translation in the future

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

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

Isolated pulmonary vasculitis associated with nilotinib use: A case report.

An association between tyrosine-kinase inhibitor therapy for chronic myeloid leukemia and vascular adverse events including peripheral arterial disease, coronary artery disease, and pulmonary hypertension has been described. We present a patient who developed isolated pulmonary artery vasculitis resulting in left pulmonary artery stenosis, in addition to left coronary artery stenosis while on nilotinib. While monitoring for cardiovascular events is important, clinicians should also recognize possible drug-induced vasculitis during chronic nilotinib therapy