A Systematic Structure-Activity Study of a New Type of Small Peptidic Transfection Vector Reveals the Importance of a Special Oxo-Anion-Binding Motif for Gene Delivery.

We discovered a new class of artificial peptidic transfection vectors based on an artificial anion-binding motif, the guanidiniocarbonylpyrrole (GCP) cation. This new type of vector is surprisingly smaller than traditional systems, and our previous work suggested that the GCP group was important for promoting critical endosomal escape. We now present here a systematic comparison of similar DNA ligands featuring our GCP oxo-anion-binding motif with DNA ligands only consisting of naturally occurring amino acids. Structure-activity studies showed that the artificial binding motif clearly outperformed natural amino acids such as histidine, lysine, and arginine. It improved the ability to shuttle foreign genetic material into cells, yet successfully mediated endosomal escape. Also, plasmids that were complexed by our artificial ligands were stabilized against cytosolic degradation to some extent. This resulted in the successful expression of plasmid information (comparable to gold standards such as polyethyleneimine). Hence, our study clearly demonstrates the importance of the tailor-made GCP anion-binding site for efficient gene transfection.

miR-542-3p exerts tumor suppressive functions in neuroblastoma by downregulating Survivin.

MicroRNAs (miRNAs) are deregulated in a variety of human cancers, including neuroblastoma, the most common extracranial tumor of childhood. We previously reported a signature of 42 miRNAs to be highly predictive of neuroblastoma outcome. One miRNA in this signature, miR-542, was downregulated in tumors from patients with adverse outcome. Reanalysis of quantitative PCR and next-generation sequencing transcript data revealed that miR-542-5p as well as miR-542-3p expression is inversely correlated with poor prognosis in neuroblastoma patients. We, therefore, analyzed the function of miR-542 in neuroblastoma tumor biology. Ectopic expression of miR-542-3p in neuroblastoma cell lines reduced cell viability and proliferation, induced apoptosis and downregulated Survivin. Survivin expression was also inversely correlated with miR-542-3p expression in primary neuroblastomas. Reporter assays confirmed that miR-542-3p directly targeted Survivin. Downregulating Survivin using siRNA copied the phenotype of miR-542-3p expression in neuroblastoma cell lines, while cDNA-mediated ectopic expression of Survivin partially rescued the phenotype induced by miR-542-3p expression. Treating nude mice bearing neuroblastoma xenografts with miR-542-3p-loaded nanoparticles repressed Survivin expression, decreased cell proliferation and induced apoptosis in the respective xenograft tumors. We conclude that miR-542-3p exerts its tumor suppressive function in neuroblastoma, at least in part, by targeting Survivin. Expression of miR-542-3p could be a promising therapeutic strategy for treating aggressive neuroblastoma.

Calcium phosphate increases the encapsulation efficiency of hydrophilic drugs (proteins, nucleic acids) into poly(d,l-lactide-co-glycolide acid) nanoparticles for intracellular delivery.

Calcium phosphate/poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles with a diameter below 200 nm, loaded with either nucleic acids or proteins, were synthesized by a water-in-oil-in-water (W1/O/W2) emulsion solvent evaporation technique. The particles were stabilized by polyvinyl alcohol (PVA) and had a negative charge (zeta potential -26 mV). By the addition of calcium phosphate into the inner aqueous phase of the W1/O/W2-emulsion, the encapsulation efficiency of siRNA was increased to 37%, of DNA to 52%, and of bovine serum albumin to 78%, i.e. by a factor of 3 to 10 compared to PLGA nanoparticles without calcium phosphate. Total loadings of 8 µg siRNA, 5 µg DNA and 280 µg fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) per mg of PLGA nanoparticles were achieved by this method. The addition of an outer layer of either chitosan or polyethyleneimine (PEI) reversed the charge of the particles (zeta potential > +30 mV) and improved the cellular uptake as well as the endosomal escape of these particles as demonstrated by confocal laser scanning microscopy. Calcium phosphate-PLGA nanoparticles loaded with DNA encoding for enhanced green fluorescent protein (eGFP-DNA) showed a good transfection efficiency for epithelial cells (HeLa). Gene silencing with HeLa cells expressing eGFP gave knockdown efficiencies of 53% for anionic nanoparticles, of 68% for chitosan-coated cationic nanoparticles, and of 89% for polyethyleneimine-coated cationic nanoparticles.

Functional characterization of novel mutations affecting survivin (BIRC5)-mediated therapy resistance in head and neck cancer patients.

Survivin (BIRC5) is an acknowledged cancer therapy-resistance factor and overexpressed in head and neck squamous cell carcinomas (HNSCC). Driven by its nuclear export signal (NES), Survivin shuttles between the nucleus and the cytoplasm, and is detectable in both cellular compartments in tumor biopsies. Although predominantly nuclear Survivin is considered a favorable prognostic disease marker for HNSCC patients, the underlying molecular mechanisms are not resolved. Hence, we performed immunohistochemical and mutational analyses using laser capture microdissection on HNSCC biopsies from patients displaying high levels of nuclear Survivin. We found somatic BIRC5 mutations, c.278T>C (p.Phe93Ser), c.292C>T (p.Leu98Phe), and c.288A>G (silent), in tumor cells, but not in corresponding normal tissues. Comprehensive functional characterization of the Survivin mutants by ectopic expression and microinjection experiments revealed that p.Phe93Ser, but not p.Leu98Phe inactivated Survivin's NES, resulted in a predominantly nuclear protein, and attenuated Survivin's dual cytoprotective activity against chemoradiation-induced apoptosis. Notably, in xenotransplantation studies, HNSCC cells containing the p.Phe93Ser mutation responded significantly better to cisplatin-based chemotherapy. Collectively, our results underline the disease relevance of Survivin's nucleocytoplasmic transport, and provide first evidence that genetic inactivation of Survivin's NES may account for predominantly nuclear Survivin and increased therapy response in cancer patients.