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.
