Periodic Reporting for period 4 - Novel asthma therapy (Biocompatible nanoparticles for T cell targeted siRNA delivery as novel asthma therapy)
Período documentado: 2020-04-01 hasta 2021-12-31
The goal of the project NOVEL ASTHMA THERAPY is to develop new therapies for the treatment of asthma that address the underlying immunologic imbalance of T-helper-cells Type 2 (Th2) responses toward allergens. We have so far addressed the problem of targeting T cells with nano-sized drug carriers that deliver small interfering DNA (siRNA). The main hurdle so far was endosomal release of the drug carriers after endocytic uptake. We have addressed this problem by conjugating melittin, a peptide of bee’s venom, to parts of our polymeric nanocarriers. Additionally, we have addressed a crucial point for clinical translation, which is the development of a relevant dosage form – in our case an inhalable powder and have optimized spray drying process parameters to obtain powders for inhalation that re-disperse into nanocarriers that can be endocytosed after delivery to the lung.
Our approach is very important for society because asthma is a chronic disease that affects over 300 million people worldwide and is neither curable nor well treated in over 5% of the patients.
The overall objectives are the development of molecular treatments that decrease Th2 responses in the lung of asthmatics, the formulation with new biodegradable nanocarriers, and the development of inhalable powders.
With the development of the biodegradable spermine-transferrin-conjugates we have concluded the synthetic part of the proposal where we have developed new nanocarriers for efficient T cell transfection. Additionally, we have developed an ex vivo model of allergic asthma in precision-cut lung slices, and we have shown therapeutic downregulation of Th2 cytokines in human lung tissue in a transferrin-dependent manner. We have identified 2 siRNA sequences that efficiently target GATA-3 with the expected downstream effects of decreasing Th2 cytokine levels. Additionally, we have developed a platform technology for spray-drying RNA nanoparticles and have confirmed their efficacy to downregulate our target gene GATA-3. Due to the long delay of the approval of the animal protocol and several pandemic-related hurdles, the final in vivo experiment will be completed in 2022. Preliminary in vivo experiments with the Tf-PEI conjugate showed trends in downregulating IL-13, but the optimized nanocarrier at an optimized dosing regimen is expected to result in significantly improved lung function and significantly decreased Th2 cytokine levels overall based on the ex vivo results obtained.
Our approach is very important for society because asthma is a chronic disease that affects over 300 million people worldwide and is neither curable nor well treated in over 5% of the patients.
The overall objectives are the development of molecular treatments that decrease Th2 responses in the lung of asthmatics, the formulation with new biodegradable nanocarriers, and the development of inhalable powders.
With the development of the biodegradable spermine-transferrin-conjugates we have concluded the synthetic part of the proposal where we have developed new nanocarriers for efficient T cell transfection. Additionally, we have developed an ex vivo model of allergic asthma in precision-cut lung slices, and we have shown therapeutic downregulation of Th2 cytokines in human lung tissue in a transferrin-dependent manner. We have identified 2 siRNA sequences that efficiently target GATA-3 with the expected downstream effects of decreasing Th2 cytokine levels. Additionally, we have developed a platform technology for spray-drying RNA nanoparticles and have confirmed their efficacy to downregulate our target gene GATA-3. Due to the long delay of the approval of the animal protocol and several pandemic-related hurdles, the final in vivo experiment will be completed in 2022. Preliminary in vivo experiments with the Tf-PEI conjugate showed trends in downregulating IL-13, but the optimized nanocarrier at an optimized dosing regimen is expected to result in significantly improved lung function and significantly decreased Th2 cytokine levels overall based on the ex vivo results obtained.
In the first period, most effort were focused on the in vitro optimization of T cell targeted nanocarriers. For optimization of parameters, polyethyleneimine (PEI) was used as polymer.
Regarding formulation, PEI was conjugated with transferrin, and transfection of primary T cells and other cell lines (Jurkat cells and MCF-7 cells), which serve as an in vitro model of transferrin receptor and Glutamyl Aminotransferase-subunit A (GATA-3) overexpressing cells, was confirmed. Gene knockdown of Glycerinaldehyde-3-phosphate-Dehydrogenase (GAPDH) as a house keeping gene was optimized and confirmed to ensure that the delivery process of siRNA works successfully before new siRNA sequences for GATA-3 knockdown were screened. Subsequently, two siRNA sequences were found that resulted in successful GATA-3 knockdown. Downstream effects of GATA-3 silencing are currently being investigated and immunological assays are being implemented. While the animal protocol at LMU was still not approved, one trainee was trained in animal procedures in my lab in Detroit, and an animal experiment with a single treatment was performed, confirming our hypothesis that multiple dosing is necessary for a profound in vivo effect. This training experiment was very valuable as the equipment was afterwards moved to Munich.
On the dosage form side, we have pursued three different approaches to obtain inhalable powders. We have used cryomilling, spray drying and spray-freeze-drying. In each case, nanocarriers were characterized before they were transferred into the powder form and after resuspension of the powders. The focus of the characterization before and after drying was most importantly the size of the individual nanocarriers that tend to agglomerate during the drying processes. Therefore, process parameters were optimized for all three methods. For all proof-of-concept experiments, bulk DNA was used. After very conducive parameters were found for spray-drying DNA nanocarriers, their biological efficacy was tested with encapsulated plasmid, and subsequently, experiments were repeated, and results compared with siRNA loaded nanocarriers. Currently, process engineering for siRNA loaded nanocarriers is being optimized to avoid loss of activity at high temperatures during the spray drying, and spray-freeze-drying is therefore focused on.
In the second period, the spray-drying method was optimized in regards to process parameters, and a platform technology was developed for converting RNA nanosuspensions into dry powders. A patent was filed (“Sprühtrocknen SiRNA”, filed in October 2020) and an important manuscript published. Additionally, biodegradable and biocompatible spermine nanocarriers were synthesized and were recently protected in a patent application ("Polyspermine als Nucleinsäurecarrier" filed in February 2022). A manuscript is currently being polished for submission. Spermine-transferrin conjugates were synthesized and optimized for siRNA encapsulation and delivery, and an ex vivo model of allergic asthma was established in human lung tissue (precision-cut lung slices). In the ex vivo model, siRNA delivery to T cells was investigated, and the most promising TF-conjugates were investigated for GATA-3 knockdown efficacy and downstream effects. Reduced levels of Th2-cytokines in a Tf-dependent manner were observed, and final results will be submitted in a manuscript shortly. After the animal protocol was finally approved, initial experiments were performed to define the dosing regimen. The final in vivo experiment is expected to be concluded before September 2022.
Except for the final optimized therapeutic in vivo gene silencing experiment, all milestones were met already, and two patents were filed (“Sprühtrocknen SiRNA”, filed in October 2020 and "Polyspermine als Nucleinsäurecarrier" filed in February 2022). Several publications were published with additional ones being submitted in 2022.
Regarding formulation, PEI was conjugated with transferrin, and transfection of primary T cells and other cell lines (Jurkat cells and MCF-7 cells), which serve as an in vitro model of transferrin receptor and Glutamyl Aminotransferase-subunit A (GATA-3) overexpressing cells, was confirmed. Gene knockdown of Glycerinaldehyde-3-phosphate-Dehydrogenase (GAPDH) as a house keeping gene was optimized and confirmed to ensure that the delivery process of siRNA works successfully before new siRNA sequences for GATA-3 knockdown were screened. Subsequently, two siRNA sequences were found that resulted in successful GATA-3 knockdown. Downstream effects of GATA-3 silencing are currently being investigated and immunological assays are being implemented. While the animal protocol at LMU was still not approved, one trainee was trained in animal procedures in my lab in Detroit, and an animal experiment with a single treatment was performed, confirming our hypothesis that multiple dosing is necessary for a profound in vivo effect. This training experiment was very valuable as the equipment was afterwards moved to Munich.
On the dosage form side, we have pursued three different approaches to obtain inhalable powders. We have used cryomilling, spray drying and spray-freeze-drying. In each case, nanocarriers were characterized before they were transferred into the powder form and after resuspension of the powders. The focus of the characterization before and after drying was most importantly the size of the individual nanocarriers that tend to agglomerate during the drying processes. Therefore, process parameters were optimized for all three methods. For all proof-of-concept experiments, bulk DNA was used. After very conducive parameters were found for spray-drying DNA nanocarriers, their biological efficacy was tested with encapsulated plasmid, and subsequently, experiments were repeated, and results compared with siRNA loaded nanocarriers. Currently, process engineering for siRNA loaded nanocarriers is being optimized to avoid loss of activity at high temperatures during the spray drying, and spray-freeze-drying is therefore focused on.
In the second period, the spray-drying method was optimized in regards to process parameters, and a platform technology was developed for converting RNA nanosuspensions into dry powders. A patent was filed (“Sprühtrocknen SiRNA”, filed in October 2020) and an important manuscript published. Additionally, biodegradable and biocompatible spermine nanocarriers were synthesized and were recently protected in a patent application ("Polyspermine als Nucleinsäurecarrier" filed in February 2022). A manuscript is currently being polished for submission. Spermine-transferrin conjugates were synthesized and optimized for siRNA encapsulation and delivery, and an ex vivo model of allergic asthma was established in human lung tissue (precision-cut lung slices). In the ex vivo model, siRNA delivery to T cells was investigated, and the most promising TF-conjugates were investigated for GATA-3 knockdown efficacy and downstream effects. Reduced levels of Th2-cytokines in a Tf-dependent manner were observed, and final results will be submitted in a manuscript shortly. After the animal protocol was finally approved, initial experiments were performed to define the dosing regimen. The final in vivo experiment is expected to be concluded before September 2022.
Except for the final optimized therapeutic in vivo gene silencing experiment, all milestones were met already, and two patents were filed (“Sprühtrocknen SiRNA”, filed in October 2020 and "Polyspermine als Nucleinsäurecarrier" filed in February 2022). Several publications were published with additional ones being submitted in 2022.
Two additional aspects were investigated that are often ignored in the field of targeted siRNA delivery. For a deeper understanding of the interaction between transferrin modified nanocarriers and the transferrin receptor, we are currently performing surface plasmon resonance spectroscopy (SPR) and microscale thermophoresis (MST) to optimize receptor binding, specificity of binding and kinetics of binding and releasing from the receptor.
The other aspect that is often neglected is endosomal release. We found, however, that most nanocarriers that are successfully endocytosed get stuck in the endosome, and subsequent gene knockdown is therefore either insufficient or unmeasurable. We have therefore additionally prepared polymer conjugates with melittin, a peptide from bee’s venom, that can successfully mediate endosomal release. We are currently optimizing the formulation in a way that encapsulates melittin in the core of the nanocarriers to avoid nonspecific toxicity.
The other aspect that is often neglected is endosomal release. We found, however, that most nanocarriers that are successfully endocytosed get stuck in the endosome, and subsequent gene knockdown is therefore either insufficient or unmeasurable. We have therefore additionally prepared polymer conjugates with melittin, a peptide from bee’s venom, that can successfully mediate endosomal release. We are currently optimizing the formulation in a way that encapsulates melittin in the core of the nanocarriers to avoid nonspecific toxicity.