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.
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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.
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 cmRNA
in 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 cmRNA
in vivo after intratracheal (i.t.) or intravenous (i.v.) injection of the assembled cmRNA
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 cmRNA
was quantified by hCFTR ELISA, and cmRNA
values were assessed by RT-qPCR. Thereby, we observed a significant improvement of lung function, especially in regards to FEV
, suggesting NP-cmRNA
as promising therapeutic option for CF patients independent of their CFTR genotype.
Cystic Fibrosis (CF) is the most common monogenic disease among people of Western European descent and caused by mutations in the CFTR gene. However, the disease severity is immensely variable even ...among patients with similar CFTR mutations due to the possible effect of 'modifier genes'. To identify genetic modifiers, we applied RNA-seq based transcriptomic analyses in CF patients with a mild and severe lung phenotype. Global gene expression and enrichment analyses revealed that genes of the type I interferon response and ribosomal stalk proteins are potential modifiers of CF related lung dysfunction. The results provide a new set of CF modifier genes with possible implications as new therapeutic targets for the treatment of CF.
Background
Multidrug resistance is a major reason for poor treatment results in advanced hepatoblastoma (HB). Several alternative treatment options are currently under investigation to improve the ...prognosis of affected patients
Aims
This study aimed to analyse the impact of sorafenib on the viability of HB cells and xenotransplanted HB tumours.
Methods
Cell viability and apoptosis were evaluated in two HB cell lines (HUH6 and HepT1) after treatment with sorafenib using MTT and Caspase 3 activation assay. Extracellular signal‐regulated kinase (ERK) phosphorylation was investigated using Western blot. In addition, sorafenib (30 mg/kg) was administered orally to NMRI mice bearing subcutaneous HUH6 derived tumours. Tumour progression and viability were monitored by tumour volume and α‐fetoprotein (AFP) levels, and apoptosis was assessed using TUNEL assay. Tumour angiogenesis and mean vascular density (MVD) was determined using CD31 staining, ERK phosphorylation was detected using indirect immunofluorescence.
Results
Treatment with sorafenib led to decreased ERK phosphorylation, reduced cell viability and induction of apoptosis in HepT1 and HUH6 cells. In HB xenografts, sorafenib significantly reduced tumour growth compared with control (P < 0.05). AFP levels were lower in the sorafenib group (P = 0.07). Relative apoptotic areas detected using TUNEL assay were increased (P = 0.003). CD31 staining revealed inhibition of angiogenesis, and mean vascular density was lower in the sorafenib group (P = 0.02). ERK phosphorylation was reduced in tumours tissues after sorafenib treatment.
Conclusion
Treatment with sorafenib led to a potent inhibition of cell viability, tumour progression and angiogenesis. Sorafenib might therefore also be a promising treatment option for high risk or recurrent HB.
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.
Treatment outcome of children with pediatric hepatocellular carcinoma (pHCC) is poor. Therefore, we evaluated the tyrosine kinase inhibitor sorafenib in a model of pHCC.
Cell viability after ...treatment with sorafenib was evaluated in HC-AFW1 cells (pHCC) using MTT assay and compared to an adult HCC (aHCC) and two hepatoblastoma (HB) cell lines. ERK, pERK, E-cadherin, and vimentin expression were investigated using Western Blot. Sorafenib (60mg/kg) was administered orally to NOD.Cg-Prkdcscid-IL2rgtmWjl/Sz mice bearing subcutaneous HC-AFW1-derived tumors. Tumor progression, viability, and vascularization were monitored by tumor volume, AFP levels, and CD31 immunostaining, respectively. Sensitization to sorafenib was evaluated using the β-catenin inhibitor ICG001.
Sorafenib reduced cell viability in HC-AFW1 (IC50: 8µM), comparable to HB cells, however less pronounced in aHCC cells (IC50: 23µM). Sorafenib inhibited ERK signaling in both, HC-AFW1 cells and -xenografts. In vivo, sorafenib treatment only led to a moderate tumor growth inhibition, although significant reduction of vascularization and tumor growth kinetics was observed. Long-term treatment with sorafenib decreased E-cadherin, but showed no induction of vimentin expression. Combining sorafenib with a β-catenin inhibitor led to an additional reduction of cell viability.
Sorafenib together with inhibitors of the β-catenin pathway might be an effective tool in the treatment of pediatric HCC.
•Sorafenib reduces viability and ERK signaling in pHCC cells.•In vivo, sorafenib exerts a moderate tumor growth inhibition.•There was a significant reduction of vascularization.•Epithelial–mesenchymal transition is not involved in the resistance to sorafenib.•Inhibition of β-catenin signaling might enhance the effect of sorafenib in pHCC.
Chemoresistance and advanced tumour stage at time of diagnosis are the major reasons for poor treatment results in hepatoblastoma (HB) and paediatric hepatocellular carcinoma (HCC). Positive results ...with transplantation of liver and bone marrow revealed the impact of the immune system on the treatment of liver malignancies. Cytotoxic-immune-cells-like natural killer (NK) and T cells are major player in the defence against developing tumours. This study aimed to specifically analyse the ability of ex-vivo expanded gamma delta T cells to recognise and lyse HB and HCC cell lines in coculture assays. Cell viability after treatment with gamma delta T cells was evaluated with two HB (HUH6 and HepT1) and one HCC cell line (HC-AFW1) using a MTT-based cytotoxicity assay. The binding of T cells to target cells was monitored using immunofluorescence microscopy. Incubation of hepatic tumour cell lines with gamma delta T cells led to a significant decrease in tumour cell viability. This was enhanced by zoledronic acid and histone deacetylase inhibitors. MT110, an EpCAM/CD3-bispecific BiTE antibody could bluntly enhance tumour cell lysis close to completion. gamma delta T cells efficiently interacted with HB and HCC cells in a spheroid culture model. Bispecific antibodies such as MT110 might be used to intensify the antitumoural effect of gamma delta T cells in context of adoptive immune cell transfer. Optimised immunotherapeutic strategies might therefore improve the outcome of high risk hepatoblastoma and hepatocellular carcinoma.
Background
Chemoresistance and advanced tumour stage at time of diagnosis are the major reasons for poor treatment results in hepatoblastoma (HB) and paediatric hepatocellular carcinoma (HCC). ...Positive results with transplantation of liver and bone marrow revealed the impact of the immune system on the treatment of liver malignancies.
Aim
Cytotoxic‐immune‐cells‐like natural killer (NK) and T cells are major player in the defence against developing tumours. This study aimed to specifically analyse the ability of ex‐vivo expanded γδ T cells to recognise and lyse HB and HCC cell lines in coculture assays.
Methods
Cell viability after treatment with γδ T cells was evaluated with two HB (HUH6 and HepT1) and one HCC cell line (HC‐AFW1) using a MTT‐based cytotoxicity assay. The binding of T cells to target cells was monitored using immunofluorescence microscopy.
Results
Incubation of hepatic tumour cell lines with γδ T cells led to a significant decrease in tumour cell viability. This was enhanced by zoledronic acid and histone deacetylase inhibitors. MT110, an EpCAM/CD3‐bispecific BiTE antibody could bluntly enhance tumour cell lysis close to completion. γδ T cells efficiently interacted with HB and HCC cells in a spheroid culture model.
Conclusion
Bispecific antibodies such as MT110 might be used to intensify the antitumoural effect of γδ T cells in context of adoptive immune cell transfer. Optimised immunotherapeutic strategies might therefore improve the outcome of high risk hepatoblastoma and hepatocellular carcinoma.
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