Investigation of saponins as novel transfection enhancer

The ability of specific triterpenoid saponins to act as a novel mediator of the endosomal release was investigated thoroughly in the past. By interacting with the endosomal membrane, these sec-ondary plant metabolites from Saponaria- or Gypsophila-specimen are able to release the endo-somal or lysosomal content before being degraded by lysosomal enzymes. Special attention hereby was paid to the use of ribosome-inactivating proteins, which can induce the apoptotic cell death by cleaving an adenine out of the 28S-Subunit of the ribosomes. A synergy between triterpenoid saponins and RIPs achieved an amplified cell toxicity in several studies, which was even tested successfully in many in-vivo-studies. Resulting out of a less specific release from the endosomes, a logic consequence was to test the ability of the triterpenoid saponins in other scien-tific fields. Therefore, the quest of this work happened to be the investigation of the saponin me-diated enhanced gene delivery (transfection). For this purpose, a number of different nanocarriers were formulated and analyzed for their suitability as stable nanoplexes in terms of size, zetapotential, uniformity, stability and DNA-complexation efficiency. After the establishment of a robust transfection method with peptide-based nanoplexes, a number of different saponins could be tested on their transfection enhancing properties, among them SO1861, isolated from Saponaria officinalis L., and GE1741, isolated from Gypsophila elegans M.Bieb. In addition, a further breakdown of the structure-activity relationship regarding the endosomal release could be achieved. After the identification of the highly active saponins SO1861 and GE1741, not only the transfection efficiency, but also the universal applicability was investigated. A flexible as-signment could be demonstrated in different cell lines, transfection methods as well as with dif-ferent nucleic acids. The fact of a not elucidated mechanism of the modulated endosomal escape was a further topic of this work. By applying microscopic and fluorescence-based methods, val-uable insights regarding the intracellular uptake and release could be gathered. With the transfec-tion of a RIP-gene the opportunity of a concrete therapeutic application was investigated. Initial in-vitro-tests confirmed a non-toxic reporter gene delivery as well as an effective and highly toxic suicide gene delivery. On neuroblastoma bearing NMRI nu/nu mice, finally an in-vivo-efficacy was demonstrated, after the combined application of SO1861 and targeted YD-nanocarrier (suicide gene) led to a 50% decrease of tumor growth. In this work, for the first time the big potential of specific triterpenoid saponins as potent trans-fection enhancer could be described. Despite detailed investigations and connected with that, an establishment of a sound basis, a further breakdown of the mechanistic actions and precise opti-mizations are highly necessary in order to manifest a big value of saponins in future gene and cancer therapies.Die Fähigkeit bestimmter Triterpensaponine als Mediator des endosomalen Release zu agieren, wurde in der Vergangenheit intensiv beforscht. Durch Interaktion mit der Endosomenmembran sind diese Sekundärstoffe aus u.a. Saponaria- oder Gypsophila-Arten in der Lage, den endoso-malen Inhalt vor dem lysosomalen Abbau aus den späten Endosomen oder Lysosomen zu befrei-en. Ein besonderes Interesse galt dabei dem Einsatz von Ribosomen-inaktivierenden Proteinen, die durch enzymatische Abspaltung eines Adenins aus der 28S-Unterheit der Ribosomen den apoptotischen Zelltod induzieren. Ein Synergismus zwischen den Triterpensaponine und RIPs verzeichnete in zahlreichen Experimenten eine vervielfachte Zytotoxizität, die gar in vielen in-vivo-Studien erfolgreich getestet wurde. Aus der weniger spezifischen Freigabe aus den Endo-somen resultierend, war es lediglich eine logische Konsequenz, die Fähigkeit der Triterpensa-ponine an weiteren Gebieten zu testen. So wurde es in dieser Arbeit zur Aufgabe, die Möglich-keiten eines durch Saponine verstärkten Gentransfers (Transfektion) zu testen. Für diesen Zweck wurden zunächst eine Reihe verschiedener Nanocarrier formuliert und durch ihre Größe, Zetapotential, Uniformität, Stabilität sowie DNA-Komplexierungseffizienz hinsicht-lich ihrer Eignung als stabile Nanoplexe analysiert. Nach der Etablierung einer robusten Trans-fektionsmethode mit peptidbasierten Nanoplexen konnte eine Vielzahl verschiedener Saponine auf ihre transfektionsverstärkenden Eigenschaften getestet werden. Darunter gehörten unter an-derem das aus Saponaria officinalis L. isolierte SO1861 und aus Gypsophila elegans M.Bieb. isolierte GE1741. Mithilfe der Screenings konnte zudem eine weitere Aufschlüsselung der bisher beschriebenen Struktur-Wirkungs-Beziehung bezüglich des endosomalen Release erreicht wer-den. Mit der Identifizierung der hochpotenten Saponine SO1861 und GE1741 wurden nicht nur die Transfektionseffizienz, sondern gleichermaßen die universellen Anwendungsmöglichkeiten untersucht. Dabei wurde ein flexibler Einsatz der Saponine hinsichtlich verschiedener Zelllinien, verschiedener Transfektionsmethoden oder gar verschiedener Nukleinsäuren demonstriert. Die Tatsache eines bisher nicht aufgeklärten Mechanismus des modulierten endosomalen Release war ferner Gegenstand dieser Arbeit. Anhand diverser mikroskopischen oder fluoreszenzbasierten Methoden konnten wertvolle Erkenntnisse hinsichtlich der intrazellulären Aufnahme sowie Freigabe gewonnen werden. Mit der Transfektion eines RIP-Gens wurde eine neue Methode der nicht viralen Tumor-Gentherapie demonstriert, sowie die Möglichkeit einer konkreten therapeutischen Anwendung erstmalig ergründet. Initiale in-vitro-Tests bestätigten hierbei einen nicht toxischen Reporter-Gentransfer sowie einen effektiven und hochtoxischen Suizid-Gentransfer. An NMRI nu/nu-Mäusen mit beimpftem Neuroblastoma wurde schließlich zum ersten Mal durch eine saponin-vermittelte in-vivo-Gentherapie eine Wirksamkeit demonstriert, nachdem durch eine kombinierte Gabe von SO1861 und gerichteten YD-Nanocarriern (Suizidgen) eine 50%ige Tumorwachs-tumshemmung beobachtet worden war. In dieser Arbeit konnte somit erstmals das große Potential spezifischer Triterpensaponine als potente Transfektionsverstärker beschrieben werden. Trotz der umfassenden Untersuchungen und der damit verbundenen Erstellung einer fundierten Wissensgrundlage ist eine weitere Erfor-schung der beschriebenen Anwendung jedoch dringend notwendig. Eine weitere Entschlüsselung der genauen mechanistischen Vorgänge sowie präzise Optimierungsschritte sind von ungemeiner Wichtigkeit, um einen hohen Wert für die zukünftige Gen- und Krebstherapie zu manifestieren

Glycosylated Triterpenoids as Endosomal Escape Enhancers in Targeted Tumor Therapies

Protein-based targeted toxins play an increasingly important role in targeted tumor therapies. In spite of their high intrinsic toxicity, their efficacy in animal models is low. A major reason for this is the limited entry of the toxin into the cytosol of the target cell, which is required to mediate the fatal effect. Target receptor bound and internalized toxins are mostly either recycled back to the cell surface or lysosomally degraded. This might explain why no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date although more than 500 targeted toxins have been developed within the last decades. To overcome the problem of insufficient endosomal escape, a number of strategies that make use of diverse chemicals, cell-penetrating or fusogenic peptides, and light-induced techniques were designed to weaken the membrane integrity of endosomes. This review focuses on glycosylated triterpenoids as endosomal escape enhancers and throws light on their structure, the mechanism of action, and on their efficacy in cell culture and animal models. Obstacles, challenges, opportunities, and future prospects are discussed

An advanced method for the small-scale production of high-quality minicircle DNA

Minicircle DNA is a promising tool in the field of gene therapy, whose products are increasingly gaining market access. Greater transfection efficiency and longer expression time as well as lower immunogenicity contrast with cost-intensive production, which also stands in the way of a broader use of the advantages of this technology in research. Starting from a commercial minicircle production kit a simple protocol for the cost-effective small-scale production of high-quality minicircle DNA to be used at a research scale has been developed by combining and improving procedures of various publications. An optimized size-exclusion chromatography method led to almost pure minicircle DNA with a superior proportion of the desired supercoiled plasmid conformation. The pharmaceutical potential of the produced minicircle DNA was investigated in vitro by real-time impedance assays in a tumor cell model in case of coded suicide genes as well as by ELISA of the translation product in case of coded human coagulation factor IX

An unusual type I ribosome-inactivating protein from Agrostemma githago L.

Agrostemma githago L. (corn cockle) is an herbaceous plant mainly growing in Europe. The seeds of the corn cockle are toxic and poisonings were widespread in the past by consuming contaminated flour. The toxic principle of Agrostemma seeds was attributed to triterpenoid secondary metabolites. Indeed, this is in part true. However Agrostemma githago L. is also a producer of ribosome-inactivating proteins (RIPs). RIPs are N-glycosylases that inactivate the ribosomal RNA, a process leading to an irreversible inhibition of protein synthesis and subsequent cell death. A widely known RIP is ricin from Ricinus communis L., which was used as a bioweapon in the past. In this study we isolated agrostin, a 27 kDa RIP from the seeds of Agrostemma githago L., and determined its full sequence. The toxicity of native agrostin was investigated by impedance-based live cell imaging. By RNAseq we identified 7 additional RIPs (agrostins) in the transcriptome of the corn cockle. Agrostin was recombinantly expressed in E. coli and characterized by MALDI-TOF-MS and adenine releasing assay. This study provides for the first time a comprehensive analysis of ribosome-inactivating proteins in the corn cockle and complements the current knowledge about the toxic principles of the plant