Periodic Reporting for period 2 - ENDOSCAPE (ENDOSCAPE, a clinically applicable non-viral gene delivery technology)
Periodo di rendicontazione: 2020-07-01 al 2021-12-31
The ENDOSCAPE technology aims to create a non-viral gene delivery technology comprising a scaffold that carries all required components, the EEE, a targeting ligand, and the effector gene. Proof of concept of the ENDOSCAPE technology will have a major impact on the therapeutic opportunities for current and future biopharmaceuticals with intracellular sites of action. Thus, overall objectives of the ENDOSCAPE project are to solve the longstanding problem of cytosolic and nucleic delivery of gene therapeutics, to minimize treatment risks by circumventing virus-mediated gene transfer, to enhance the efficacy of targeted gene therapeutic treatment in patients, to reduce the costs of gene therapy and make it available for a broad patient base, and to be compatible with personalized gene therapies.
After developing conditions to complex example effector genes, we analyzed a large panel of complete ENDOSCAPE prototypes consisting of a scaffold, EEEs, targeting ligand and the gene. To ensure the quality, 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 then continued with the functional characterization of the ENDOSCAPE prototypes in cell cultures and investigated their molecular mode of action. Here we first worked with the gene of enhanced green fluorescent protein as a reporter and surrogate for the therapeutic genes applied in the project for hemophilia therapy and suicide genes for cancer treatment. We further refined recombinant expression and purification of several candidate ligands and were able to select the most performing scaffolds and ligands to target hepatoma and cancer cells. To test toxicity and stability of the prototypes in human blood, we developed new assays for ex vivo measurements and have created the conditions for mouse experiments that have just started. 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 a post-harvest treatment 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 with the intention to manipulate the biosynthesis of EEEs for higher yields. An extraction and purification protocol for highly pure EEEs and related compounds was established.
In addition, we continued the early health economic evaluation by defining clinical example applications and conducting analyses based on literature review and expert inputs. This 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. Our project is regularly communicated, e.g. by a public website, a project video, and a LinkedIn account.