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The technical importance of paraffins as phase change materials (PCM) in heat storage systems increases. Knowledge on the thermal conductivity of paraffins is necessary for the design and optimization of heat storage systems. However, for most paraffins solely the thermal conductivity of the liquid state has been sufficiently investigated. For the solid state, precise thermal conductivity data are only known for a few paraffins, while only generalized values are available for the remainder, some of which contradict each other. In this study, a measurement setup based on the modified guarded hot plate method is developed. It is used to investigate the thermal conductivity of several paraffines in the solid state, including pure n-docosane and its compounds with different types and concentrations of graphite. For n-docosane in the solid state, the thermal conductivity is determined to be 0.49 W m−1 K−1. A particle size of 200 μm with a spherical shape turns out to be optimal to increase the thermal conductivity. This allows the thermal conductivity of a compound with 10% graphite to increase by a factor of three compared to the pure paraffin. Furthermore, significant differences to thermal conductivity data from the literature are found.

There exists a dire need for improved therapeutics to achieve predictable bone regeneration. Gene therapy using non-viral vectors that are safe and efficient at transfecting target cells is a promising approach to overcoming the drawbacks of protein delivery of growth factors. Here, we investigated the transfection efficiency, cytotoxicity, osteogenic potential and in vivo bone regenerative capacity of chemically modified ribonucleic acid (cmRNA) (encoding BMP-2) complexed with polyethylenimine (PEI) and made comparisons with PEI complexed with conventional plasmid DNA (encoding BMP-2). The polyplexes were fabricated at an amine (N) to phosphate (P) ratio of 10 and characterized for transfection efficiency using human bone marrow stromal cells (BMSCs). The osteogenic potential of BMSCs treated with these polyplexes was validated by determining the expression of bone-specific genes, osteocalcin and alkaline phosphatase as well as through the detection of bone matrix deposition. Using a calvarial bone defect model in rats, it was shown that PEI-cmRNA (encoding BMP-2)-activated matrices promoted significantly enhanced bone regeneration compared to PEI-plasmid DNA (BMP-2)-activated matrices. Our proof of concept study suggests that scaffolds loaded with non-viral vectors harboring cmRNA encoding osteogenic proteins may be a powerful tool for stimulating bone regeneration with significant potential for clinical translation.

Employing cost-effective biomaterials to deliver chemically modified ribonucleic acid (cmRNA) in a controlled manner addresses the high cost, safety concerns, and lower transfection efficiency that exist with protein and gene therapeutic approaches. By eliminating the need for nuclear entry, cmRNA therapeutics can potentially overcome the lower transfection efficiencies associated with non-viral gene delivery systems. Here, we investigated the osteogenic potential of cmRNA-encoding BMP-9, in comparison to cmRNA-encoding BMP-2. Polyethylenimine (PEI) was used as a vector to increase in vitro transfection efficacy. Complexes of PEI-cmRNA (encoding BMP-2 or BMP-9) were fabricated at an amine (N) to phosphate (P) ratio of 10 and characterized for transfection efficacy in vitro using human bone marrow stromal cells (BMSCs). The osteogenic potential of BMSCs treated with these complexes was determined by evaluating the expression of bone-specific genes as well as through the detection of bone matrix deposition. It was found that alkaline phosphatase (ALP) expression 3 days post transfection in the group treated with BMP-9-cmRNA was significantly higher than that in the group that received BMP-2-cmRNA treatment. Alizarin red staining and atomic absorption spectroscopy demonstrated enhanced osteogenic differentiation as evidenced by increased bone matrix production by the BMSCs treated with BMP-9-cmRNA when compared to cells treated with BMP-2-cmRNA. In vivo studies showed increased bone formation in calvarial defects treated with the BMP-9-cmRNA and BMP-2-cmRNA collagen scaffolds when compared to empty defects. The connectivity density of the regenerated bone was higher (2-fold-higher) in the group that received BMP-9-cmRNA compared to BMP-2-cmRNA. Together, these findings suggest that cmRNA-activated matrix encoding osteogenic molecules can provide a powerful strategy for bone regeneration with significant clinical translational potential.

Genome editing using site-specific endonucleases (SSE) holds great potential to repair disease-causing genes. In this study we offer a promising approach to deliver SSEs as nuclease-encoded, chemically modified mRNA (nec-mRNA) together with a repair template to correct the lung disease Surfactant Protein-B (SP-B) deficiency while simultaneously reducing SSE-induced off-target effects. To assess the potential of mRNA-encoded nucleases to create site specific double-strand breaks (DSBs), we evaluated a panel of 15 transcription activator-like effector nucleases (TALENs) and 36 Zink-Finger nucleases (ZFNs) for non-homologous end-joining (NHEJ) and also tested for the integration of a promoter template by homology-directed repair (HDR) in vitro . In addition we compared different mRNA modifications to assess immunoreactions against nec-mRNA. Finally, we co-administered intratracheally nec-mRNA with the repair template to SP-B deficient mice and evaluated the survival and promoter integration efficacy. Compared to plasmid-DNA, delivery of mRNA-encoded nucleases resulted in more frequent induction of DSBs ( P P in vitro . While being only transiently expressed, incorporation of modified nucleosides into nec-mRNA and complexation into nanoparticles (NPs) increased mRNA expression levels ( P in vivo . Also, no immune activation was observed. Importantly, delivery of nec-mRNA-NP to SP-B deficient mice lead to prolonged survival rates compared with matched control groups ( P In conclusion, we have shown that delivery of nec-mRNA-NP together with a repair template results in successful, site-specific genome editing in vivo .

The response of standard-risk hepatoblastoma (HB) to neoadjuvant cisplatin (CDDP) chemotherapy is excellent; however, in high-risk HB, drug resistance remains a major challenge. Alternative therapeutic strategies may consider combining cytotoxic drugs with apoptosis sensitizers as this has shown additive effects in various types of malignancies. Analysis of published expression databases have revealed an anti-apoptosis state in HB samples. Herein, we evaluated the synergistic effects of ABT-737 as a modulator of apoptosis in combination with CDDP in HB. To this end, clonogenic assays were performed with HepT1 and HUH6 HB cells to evaluate the synergistic effects of CDDP and ABT-737. Combination treatment with CDDP and ABT-737 reduced the clonogenicity of HB cells more than 5-fold compared to treatment with CDDP alone. Furthermore, the HUH6 mixed-type HB cells showed higher sensitivity to CDDP and combination treatment compared to the HepT1 embryonal-type cells. Subcutaneous HUH6 tumors in NOD/LtSz-scid IL2Rγnull mice were treated with CDDP (1.25 and 3 mg/kg body weight, n=6), ABT-737 (100 mg/kg, n=5) and the combination of both agents (n=5). Combined treatment led to a significantly reduced tumor growth compared to CDDP treatment alone (p

Zink-Finger- bzw. transcription activator-like effector-Nukleasen (ZFNs/TALENs) sind vielversprechende Werkzeuge im Zuge der Reparatur genomischer Mutationen, indem diese den Mechanismus der Homologen Rekombination begünstigen. In Studien zur Hämophilie konnte bereits eine Nuklease-vermittelte Genkorrektur unter Verwendung von AAV-Vektoren demonstriert werden. Dabei kodierten diese sowohl für die Doppelstrangbruch- (DSB) induzierenden ZFN-Nukleasen, als auch den Einbau des Reparatur-Templates durch den Reparaturmechanismus der Homologen Rekombination (HDR, engl. homology-directed repair). Während der Einsatz von stabil exprimierenden AAV-Vektoren zwar als sicher angesehen werden kann, ist eine transiente Expression von Nukleasen ausreichend, um genomische Modifikationen zu erzeugen. Demzufolge wäre die zeitlich begrenzte Expression von ZFNs ein entscheidender Vorteil, um potentielle Nuklease-bedingte Nebeneffekte zu reduzieren. In den letzten Jahren hat sich als Alternative zu traditionellen, viralen gentherapeutischen Vektoren, modifizierte messenger RNA (mRNA) als vielversprechend herausgestellt. Dessen Vorteile liegen sowohl in einer transienten Proteinexpression, bei gleichzeitiger Vermeidung genomischer Integration, als auch in dessen chemischen Modifizierungen, die eine stabile Expression und das Ausbleiben von Immunantworten begünstigen. Eine solche Co-Transfektion mRNA-kodierender Nukleasen in einem gentherapeutischen Kontext macht mRNA daher zu einem idealen Vehikel. In dieser Studie wurden solche optimierten mRNA-kodierenden Nukleasen verwendet, um die erfolgreiche Genkorrektur am murinen Modell der SP-B Defizienz zu demonstrieren. Für die Genkorrektur des SP-B Lokus wurde zunächst ein Panel aus in silico generierten ZFNs und TALENs hergestellt. Der dual-luciferase single-strand Assay sowie der T7-Endonuklease I -Assay verifizierten für TALEN-1 (T1) und ZFN-3 (Z3) eine hohe Schnitt-Effizienz ex vivo, wodurch bis zu 39 % der Allele durch das nicht-homologe end-joining (NHEJ) repariert wurden. Im Gegensatz zu Plasmid-DNA (pDNA) stieg dabei die Induktion an DSBs signifikant an (P < 0,05). Die Kombination mit einem Reparatur-Template, welches eine NheI-Restriktionsschnittstelle in den SP-B Lokus einfügte, konnte ebenfalls ein signifikanter Anstieg in der HDR für mRNA verzeichnet werden (P < 0,05). Da Z3 effizienter in der DSB-Induktion sowie der HDR war, wurde diese für alle folgenden Experimente verwendet. Zur Optimierung der Z3-Expression in vivo wurden zwei Modifikationen (Ψ(1.0)/m5C(1.0) und s2U(0.25)/m5C(0.25)) mit und ohne Komplexierung in mit Chitosan-beschichteten poly(D,L-Laktid-Co-Glycolid) Nanopartikeln (NPs) getestet. Dabei ergaben sich für markierte s2U(0.25)/m5C(0.25) Z3 mRNA (im Weiteren als Z3 nec-mRNA bezeichnet) mit NPs die höchsten Expressionslevel- nach intratrachealer (i.t.) Applikation in die Lunge. Über einen IFN-α ELISA wurde zusätzlich ein signifikanter immunstimulatorischer Effekt der Z3 nec-mRNA ausgeschlossen. Die sequenzspezifische HDR wurde anschließend ex vivo über die Co-Applikation eines CAG-Promoter enthaltenden AAV- Donor-Templates (AAV6-Donor) zusammen mit AAV-kodierender Z3 (Z3 AAV) oder Z3 nec-mRNA erfolgreich getestet. Diese gentherapeutische Manipulation zeigte sich ebenfalls in Gen-korrigierten, transgenen SP-B Mäusen mit signifikant höheren Überlebensraten als die der Mock-behandelten Gruppen (P < 0,001). Korrigierte SP-B Mäuse zeigten dabei eine normale Lungenfunktion im Vergleich zu Positivkontrollen. Ebenfalls wurde im Vergleich zu PBS-behandelten Mäusen kein signifikanter Anstieg in der IL-12 Produktion beobachtet. Sequenzspezifische DSB-Induktion sowie HDR bestätigten die Ausprägung eines normalen Phänotyps, wobei zwischen Z3 AAV und Z3 nec-mRNA keine Unterschiede zu erkennen waren. Die immunhistochemischen Analysen bestätigten zusätzlich die zeitlich limitierte Expression der Z3 nec-mRNA im Vergleich zu Z3 AAV. Die hier erhaltenen Ergebnisse bestätigen eindrucksvoll, dass die Applikation von nec-mRNA in Kombination eines AAV-Donors zur erfolgreichen Genkorrektur des transgenen SP-B Lokus führt und somit die konstitutive SP-B Expression sowie eine normale Lungenfunktion nach Absetzen von Doxycyclin erlaubt. Verglichen mit AAV, zeigte Z3 nec-mRNA eine zeitlich limitierte Nuklease-Expression, wodurch mögliche Nebeneffekte im Gegensatz zu dauerhaft aktiven AAV-Nukleasen reduziert werden können. Zusätzlich macht die Abwesenheit einer Immunantwort nec-mRNA zu einem idealen Vehikel für mehrmalige Applikationen hinsichtlich einer dauerhaften Genkorrektur der Lunge. Zusammengefasst besitzt nec-mRNA damit ein enormes Potential im Zuge klinisch relevanter, gentherapeutischer Strategien für die Behandlung erblich-bedingter Lungenerkrankungen sowie weiterer monogenetischer Defekte.

Background:Sorafenib has recently been shown to reduce tumour growth in hepatoblastoma (HB) xenografts. The effect of a combined administration with cytostatic agents was now investigated.Methods:Cell viability after treatment with sorafenib and different cytostatic agents was evaluated in two HB cell lines (HUH6 and HepT1) using MTT assay. ERK signalling was investigated by western blot, NOXA expression by rt-PCR, and formation of DNA adducts using immunocytology. NMRI mice bearing subcutaneous HUH6-derived tumours were treated with sorafenib alone or in combination with cisplatin. Tumour progression, viability, apoptosis, and vascularisation were monitored by tumour volume, AFP levels, TUNEL assay, and CD31 immunostaining, respectively.Results:The combination of sorafenib and cisplatin led to a remarkable decrease in cell viability. The cisplatin-induced enhanced ERK1/2 activation, but not NOXA expression and the formation of DNA adducts was partly abrogated by sorafenib. In HB xenografts, both, sorafenib and alternated application of sorafenib and cisplatin significantly reduced tumour growth (P

Multidrug resistance (MDR) is a major reason for poor treatment results in hepatoblastoma (HB). The objective of this study was to establish a drug resistance model for HB to analyse alternative treatment options in vitro. Both HB cell lines HUH6 and HepT1 were xenotransplanted in NMRI mice (nu/nu) and 2 cycles of cisplatin (CDDP) treatment were administered. Thereafter, xenotransplants were excised and viable tumour cells were re-cultured. 3D cultures of HUH6 and HepT1 cells were generated on a low binding culture surface. Cell viability in response to CDDP/DOXO (doxorubicin) and apoptosis was assessed by MTT-assay and caspase 3 activity, respectively. Efflux of doxorubicin was measured by flow cytometry. Cellular levels of ABC-transporters (MDR1, MRP1, cMOAT and BRCP) were determined by real time rt-PCR. Only HepT1 cells isolated from HB xenografts showed resistance to CDDP, but did not survive repeated passages. Culturing HUH6 and HepT1 cells as spheroids was successful and 3D cultures showed an IC50-drift to higher drug concentrations for CDDP and DOXO compared to 2D cultures. Treatment with CDDP and DOXO led to homogeneous apoptosis in spheroids. Increased doxorubicin efflux in HUH6 spheroids was not influenced by the P-glycoprotein inhibitor tariquidar. Expression levels of MDR1, MRP1, cMOAT and BRCP in 3D cultures were similar to those in 2D cultures and were higher in HepT1 than in HUH6 cells. In conclusion, a 3D cell culture model for multidrug resistance was established for hepatoblastoma. The underlying mechanism involves altered accessibility of the cells for drugs rather than up-regulation of ABC-transporters.

Background: Hepatoblastoma (HB) represents the most common malignant liver tumour in childhood. For patients with high risk or recurrent HB treatment results remain still poor due to a developing multidrug resistance during chemotherapy. Therefore new therapy strategies have to be evaluated. The primary objective was to investigate the combined therapy of cytotoxic agents with sorafenib. Sorafenib has been shown to reduce tumour progression and angiogenesis. In order to investigate a treatment close to a clinical setting the effect of the combined administration of cytotoxic agents with sorafenib on HB cell lines and xenotransplanted HB tumours was determined. Methods: Cell viability of two HB cell lines (HUH6 and HepT1) was evaluated in using MTT assays after treatment with sorafenib and different cytostatic agents (cisplatin (CDDP), doxorubicin, irinotecan, topotecan). ERK signalling was investigated by Western blot analysis. NMRI mice (nu/nu) bearing subcutaneous HUH6 derived tumours were treated with Sorafenib orally (30 mg/kg, on days 4-13 and 17-24) alone or in combination with CDDP i.p. (3 mg/kg, on days 1-3 and 14-16). Tumor progression and viability were monitored by tumour volume and AFP levels. Apoptosis was assessed by TUNEL assay. Angiogenesis was determined by CD31 staining and mean vascular density (MVD) was calculated. Results: In vitro the combination of sorafenib with CDDP showed a remarkable decrease in cell viability compared to the other agents tested. However with increasing concentrations of CDDP this additive effect disappeared. CDDP alone showed an enhanced phosphorylation of ERK1/2, which is a common pathway for apoptosis induction through NOXA. CDDP in combination with sorafenib led to an overall reduced phosphorylation level of ERK1/2, which also resulted in a low HB cell viability. Since CDDP and sorafenib have an opposing effect on phosphorylation of ERK1/2, we avoid the concomitant administration of both drugs in vivo. In HB xenografts both sorafenib alone or in alternating combination with CDDP reduced tumour growth compared to the control untreated group (p < 0.05). The combination treatment showed a higher and earlier reduction of tumor volume than sorafenib alone. In addition AFP levels did not significantly change in the treatment groups. Apoptotic areas as determined by TUNEL analysis increased significantly after administration of sorafenib or sorafenib and CDDP (p < 0.08). Furthermore CD31 staining showed a significant inhibition of angiogenesis as revealed by determination of the MVD in both treatment groups (p < 0.02). Discussion: Taken together the combination therapy of Sorafenib and CDDP led to an enhanced decrease in cell viability as well as in tumour progression and angiogenesis than sorafenib alone. Therefore a combined therapy might be a promising option in the treatment of high risk or recurrent hepatoblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4337. doi:1538-7445.AM2012-4337

Background The immunogenicity and limited stability of conventional messenger RNA (mRNA) has traditionally restricted its potential therapeutic use. In 1992, the first clinical application of mRNA was reported as a potential protein-replacement therapy; however, subsequent investigations have not been made for almost two decades. Recent developments, including increased stability, controlling immunogenicity, as well as utilization of mRNA encoding zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9, have implicated modified mRNA as a very promising option for cancer immunotherapy, vaccines, protein expression replacement, and genome editing. This review aims to offer a summary of our present understanding of and improvements in mRNA-based drug technologies, along with a focus on the role in therapeutic options for pediatric respiratory diseases and hemoglobinopathies. Conclusions This mini review summarizes the recent advances in modified mRNA-based therapy and its potential therapeutic effect in treating major pediatric diseases.

Gene therapy has always been a promising therapeutic approach for Cystic Fibrosis (CF). However, numerous trials using DNA or viral vectors encoding the correct protein resulted in a general low efficacy. In the last years, chemically modified messenger RNA (cmRNA) has been proven to be a highly potent, pulmonary drug. Consequently, we first explored the expression, function and immunogenicity of human (h)CFTR encoded by cmRNAhCFTRin vitro and ex vivo, quantified the expression by flow cytometry, determined its function using a YFP based assay and checked the immune response in human whole blood. Similarly, we examined the function of cmRNAhCFTRin vivo after intratracheal (i.t.) or intravenous (i.v.) injection of the assembled cmRNAhCFTR together with Chitosan-coated PLGA (poly-D, L-lactide-co-glycolide 75:25 (Resomer RG 752 H)) nanoparticles (NPs) by FlexiVent. The amount of expression of human hCFTR encoded by cmRNAhCFTR was quantified by hCFTR ELISA, and cmRNAhCFTR values were assessed by RT-qPCR. Thereby, we observed a significant improvement of lung function, especially in regards to FEV0.1, suggesting NP-cmRNAhCFTR as promising therapeutic option for CF patients independent of their CFTR genotype.