Periodic Reporting for period 3 - ENDOSCAPE (ENDOSCAPE, a clinically applicable non-viral gene delivery technology)
Período documentado: 2022-01-01 hasta 2023-06-30
Gene therapy is one of the most promising options for future advanced treatments in a broad range of diseases. Clinical delivery of gene therapeutics is currently accomplished via viral vectors, which still have major safety concerns and involve complex and costly manufacturing procedures. Typically, viral-based approaches trigger an immune response due to the induction of neutralizing antibodies and thus re-treatment is not an option at present.
In currently available non-viral technologies, more than 98% of the therapeutic genes accumulate inside cellular compartments called endosomes where they are finally degraded. In recent years, we discovered certain glycosylated triterpenoids as Endosomal Escape Enhancers (EEEs), which plants have evolved as a self-defense mechanism against pathogens. We are now adapting these EEEs to improve the delivery outcome of targeted, non-viral gene therapy. EEEs specifically accumulate in endosomal membranes, prevent gene degradation by enabling endosomal escape, and provide sufficient amount of the therapeutic gene to localize in the nucleus. However, to date such EEEs and the gene therapeutic product must be applied as two independent components, which makes clinical applicability and marketing approval complicated.
The ENDOSCAPE technology aimed at creating 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 were 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.
In conclusion, we were able to achieve most of our objectives. We created new molecular scaffolds, equipped them with EEEs and modified them with polyethylene glycol linkers to finally bind ligands. We polyplexed effector DNA and demonstrated gene transfer in vitro and in vivo. We further established a larger scale production of EEEs. Targeting of the polyplexes to target cells or organs is still a challenge that requires further optimization of the technology.
In currently available non-viral technologies, more than 98% of the therapeutic genes accumulate inside cellular compartments called endosomes where they are finally degraded. In recent years, we discovered certain glycosylated triterpenoids as Endosomal Escape Enhancers (EEEs), which plants have evolved as a self-defense mechanism against pathogens. We are now adapting these EEEs to improve the delivery outcome of targeted, non-viral gene therapy. EEEs specifically accumulate in endosomal membranes, prevent gene degradation by enabling endosomal escape, and provide sufficient amount of the therapeutic gene to localize in the nucleus. However, to date such EEEs and the gene therapeutic product must be applied as two independent components, which makes clinical applicability and marketing approval complicated.
The ENDOSCAPE technology aimed at creating 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 were 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.
In conclusion, we were able to achieve most of our objectives. We created new molecular scaffolds, equipped them with EEEs and modified them with polyethylene glycol linkers to finally bind ligands. We polyplexed effector DNA and demonstrated gene transfer in vitro and in vivo. We further established a larger scale production of EEEs. Targeting of the polyplexes to target cells or organs is still a challenge that requires further optimization of the technology.
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.
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.
The ENDOSCAPE technology aims to achieve a higher efficacy of non-viral targeted gene therapy using EEEs, and to allow for any macromolecule to be delivered to target cells, in particular to treat monogenetic diseases and cancer. ENDOSCAPE aims to provide an alternative to viral gene delivery technology while incorporating already existing medical drug candidates. ENDOSCAPE has the potential to allow the resurrection of drugs that clinically failed due to insufficient intracellular drug delivery and will strengthen the EU's competitive landscape in the global search for new advanced technologies. A positive outcome from a first in human study would accelerate the development of a broader pipeline with new ENDOSCAPE gene therapies for other disease indications, attracting new investors as well as out-licensing or co-development options with pharmaceutical or biotech companies. The ENDOSCAPE technology putatively can also be implemented for other therapeutic modalities extending the potential of the technology, broadening the field of therapeutic applications, and strongly impacting the value of the technology.