We were in constant exchange within a joint collaboration framework. This framework included a joint data server and common policies for structured and joint actions such as a Data Management Plan, a Publication Policy, and a Dissemination and Exploitation Plan. In addition, we had regular discussions with our Intellectual Property Rights and Exploitation Board and an Advisory Committee. We further held regular meetings of the whole consortium, the work package leaders, and selected working groups.
We analyzed a large panel of complete ENDOSCAPE prototypes consisting of a scaffold, EEEs, targeting ligand and the gene. ENDOSCAPE prototypes were analyzed by methods such as mass spectrometry, nuclear magnetic resonance, dynamic light scattering and fluorogenic detection, which provided feedback for further optimization cycles. The first ENDOSCAPE prototypes have been produced and chemically optimized before testing in vitro. We characterized them in cell cultures and investigated their molecular mode of action. Here we first worked with the coding sequence of enhanced green fluorescent protein as a reporter and surrogate for the therapeutic genes applied for hemophilia therapy and suicide genes for cancer treatment. We further refined recombinant expression and purification of candidate ligands and selected the most performing scaffolds and ligands to target hepatoma and cancer cells. We tested toxicity and stability of the prototypes in human blood and demonstrated in mouse studies low toxicity and low immunogenicity of the prototypes making them tolerable drugs. For dendrimer-based scaffolds, we provided evidence that the ENDOSCAPE prototypes are suitable to successfully treat mouse tumors by intratumoral injections, but to date we were not able to address the target organs by ligands placed on the surface of the prototypes, most likely due to the increased size of the polyplexes after attachment of the ligands. To understand the molecular mechanism of the escape of nucleic acids from endosomes in living cells we used fluorescently labeled ENDOSCAPE modules and confocal microscopy for a subcellular analysis of their trafficking in hepatoma cells and primary hepatocytes.
The use of plant natural products such as EEEs entails the risk of heterogeneous source material and lack of availability. Therefore, an independent production process is vitally important. We selected and optimized different plant growth systems and culturing conditions including post-harvest treatments of roots to increase the EEE quantity. We further conducted an expression analysis in EEE producing plants and discovered genes involved in the biosynthesis of EEEs. We proved that the expression product of one gene in particular is capable of boosting EEE production in root cultures of different plant species. An extraction and purification protocol for highly pure EEEs and related compounds was established.
An early health economic evaluation included the definition of a production model for a Cost of Goods analysis and of cost-effectiveness model structures, clarification of comparator treatments and relevant dimensions of ENDOSCAPE-delivered therapies and documentation for two exemplary indications, acute lymphocytic B-cell leukemia, and hemophilia B. More extensive functional assays in cell cultures or organoids as well as toxicity, pharmacokinetic and pharmacodynamic studies in animal models will need to be conducted to provide a strong preclinical data set in preparation for toxicity studies and clinical trials. First clinical studies could be initiated in 2028 with the first ENDOSCAPE non-viral gene therapy product for the treatment of hemophilia B. Our project is regularly communicated, e.g. by a public website, a project video, and a LinkedIn account.
