Periodic Reporting for period 1 - RNhale (Dry Powder Formulation of RNA Nanoparticles for Inhalation and Improved Storage and Transport Conditions)
Reporting period: 2022-07-01 to 2023-12-31
While existing RNA drugs primarily target the liver, the lung presents a variety of currently untargeted diseases for RNA therapeutics. By employing local pulmonary delivery of RNA nanoparticles, there is potential to expand drug delivery beyond the liver. Utilizing dry powder formulations for administering RNA drugs offers numerous advantages over liquid formulations including enhanced physical, chemical, and microbial stability of RNA.
The objective of the proposed ERC PoC project was to develop a platform technology for spray drying a variety of RNA LNPs containing nucleic acids with different topologies. The solution, known as RNhale, is a dry powder platform technology that produces inhalable Nanoparticles embedded in Microparticles (NEM) for pulmonary delivery. This approach offers three key advantages over currently available and marketed RNA medicines: 1) direct access to the lungs through dry powder inhalation, 2) continuous manufacturing through spray drying, and 3) physicochemical stabilization in their dry state.
The objective of the proposed ERC PoC project was to develop a platform technology for spray drying a variety of RNA LNPs containing nucleic acids with different topologies. The solution, known as RNhale, is a dry powder platform technology that produces inhalable Nanoparticles embedded in Microparticles (NEM) for pulmonary delivery. This approach offers three key advantages over currently available and marketed RNA medicines: 1) direct access to the lungs through dry powder inhalation, 2) continuous manufacturing through spray drying, and 3) physicochemical stabilization in their dry state.
1. Platform technology RNhale
Over the course of the project duration, a process was developed for the formulation of LNPs which were subsequently dialyzed, upconcentrated and after the addition of excipients transferred into NEMs via spray drying.
For the purpose of the PoC, various LNPs were produced using the automated Knauer Nanoscaler device, based on the Onpattro formulation with different lipid compositions, siRNA (NC, eGFP, GATA3) and mRNA (eGFP) cargos. Additionally, a variety of excipients were tested for optimal pulmonary delivery.
2. NEM efficiency in vitro and ex vivo
In vitro eGFP protein downregulation:
In order to evaluate the in vitro gene silencing efficiency of spray dried LNPs, they were assessed in H1299-eGFP cells, a lung carcinoma cell line with stably enhanced green fluorescent protein (eGFP) expression.
In vitro eGFP mRNA expression:
In order to evaluate the in vitro mRNA expression efficiency of spray dried LNPs, they were assessed in H1299 cells, a lung carcinoma cell line via flow cytometry and EVOS.
Ex vivo GATA3 transcription factor downregulation and toxicity evaluation:
In a model of asthmatic disease, the transcription factor GATA 3 was silenced and checked for toxic events.
3. Inhalable dry powder and powder characterization
Mass median diameter (MMD) and Fine particle fraction (FPF):
The dry powder formulations were evaluated for their inhalability using laser diffraction with an included inhaler module. A mass median diameter (MMD) between 1-5 µm is targeted. The percentage of powder particles falling within this size range is referred to as the Fine Particle Fraction (FPF).
Scanning electron microscopy (SEM):
The morphology and geometric diameter were evaluated via SEM.
Residual moisture:
The residual moisture of the dry powder formulations was evaluated by Karl-Fisher-Titration.
4. Stability
Evaluation of the dry powder properties concerning gene silencing efficiency and residual moisture content after 18 months stored at room temperature and 4 °C.
Over the course of the project duration, a process was developed for the formulation of LNPs which were subsequently dialyzed, upconcentrated and after the addition of excipients transferred into NEMs via spray drying.
For the purpose of the PoC, various LNPs were produced using the automated Knauer Nanoscaler device, based on the Onpattro formulation with different lipid compositions, siRNA (NC, eGFP, GATA3) and mRNA (eGFP) cargos. Additionally, a variety of excipients were tested for optimal pulmonary delivery.
2. NEM efficiency in vitro and ex vivo
In vitro eGFP protein downregulation:
In order to evaluate the in vitro gene silencing efficiency of spray dried LNPs, they were assessed in H1299-eGFP cells, a lung carcinoma cell line with stably enhanced green fluorescent protein (eGFP) expression.
In vitro eGFP mRNA expression:
In order to evaluate the in vitro mRNA expression efficiency of spray dried LNPs, they were assessed in H1299 cells, a lung carcinoma cell line via flow cytometry and EVOS.
Ex vivo GATA3 transcription factor downregulation and toxicity evaluation:
In a model of asthmatic disease, the transcription factor GATA 3 was silenced and checked for toxic events.
3. Inhalable dry powder and powder characterization
Mass median diameter (MMD) and Fine particle fraction (FPF):
The dry powder formulations were evaluated for their inhalability using laser diffraction with an included inhaler module. A mass median diameter (MMD) between 1-5 µm is targeted. The percentage of powder particles falling within this size range is referred to as the Fine Particle Fraction (FPF).
Scanning electron microscopy (SEM):
The morphology and geometric diameter were evaluated via SEM.
Residual moisture:
The residual moisture of the dry powder formulations was evaluated by Karl-Fisher-Titration.
4. Stability
Evaluation of the dry powder properties concerning gene silencing efficiency and residual moisture content after 18 months stored at room temperature and 4 °C.
During the 18-month project duration of RNhale, we were able to establish a reliable platform technology for the production of NEM formulations that can be administered pulmonarily as dry powder. A semi-automated process was developed, culminating in a continuous spray drying procedure. It was successfully demonstrated that a variety of LNP formulations with different lipid compositions, containing diverse RNA cargos (siRNA and mRNA), were successfully embedded in increasing concentrations in excipient matrices with excellent properties for pulmonary delivery. The goal of reaching a human dose concentration of 10% RNA in dry powder was reached.
Consistent gene silencing capability was demonstrated across various in vitro and ex vivo models compared with fresh LNP suspensions (Figure 2). Additionally, in the ex vivo model using human precision-cut lung slices (hPCLS), no toxic side effects were observed.
Furthermore, LNPs containing mRNA were successfully spray-dried. This poses a significantly greater challenge compared to siRNA, as mRNA is extremely sensitive to higher temperatures and stress that may occur during the process. All in vito and ex vivo models were carefully chosen for the sole purpose of mimicking pulmonary conditions such as cell types and pulmonary disease models.
Additionally, formulations were developed that exhibit optimal powder properties for inhalation, with a mass median diameter (MMD) ranging from 1 to 5 µm and a high fine particle fraction (FPF) exceeding 60 %. A residual moisture content of less than 7% is crucial for both good storage stability and low susceptibility to microbial contamination of the formulations. This threshold was significantly undershot in both fresh spray dried formulations and after storage at room temperature and 4 °C for 3 and 18 months (Figures 3 and 4).
In vivo and ex vivo experiments using spray dried powders confirmed therapeutically relevant GATA-3 knockdown with subsequent downregulation of Th2 cytokines and reduced eosinophilia. In human precision-cut lung slices (hPCLS), T cells were activated for three days and transfected for two days. GATA3 mRNA levels were assessed using RT-qPCR. Additionally, a multiplex ELISA was performed on the supernatants of hPCLS to evaluate IL-6, TNF-α, IL-1ß, and IL-10 activation.
Consistent gene silencing capability was demonstrated across various in vitro and ex vivo models compared with fresh LNP suspensions (Figure 2). Additionally, in the ex vivo model using human precision-cut lung slices (hPCLS), no toxic side effects were observed.
Furthermore, LNPs containing mRNA were successfully spray-dried. This poses a significantly greater challenge compared to siRNA, as mRNA is extremely sensitive to higher temperatures and stress that may occur during the process. All in vito and ex vivo models were carefully chosen for the sole purpose of mimicking pulmonary conditions such as cell types and pulmonary disease models.
Additionally, formulations were developed that exhibit optimal powder properties for inhalation, with a mass median diameter (MMD) ranging from 1 to 5 µm and a high fine particle fraction (FPF) exceeding 60 %. A residual moisture content of less than 7% is crucial for both good storage stability and low susceptibility to microbial contamination of the formulations. This threshold was significantly undershot in both fresh spray dried formulations and after storage at room temperature and 4 °C for 3 and 18 months (Figures 3 and 4).
In vivo and ex vivo experiments using spray dried powders confirmed therapeutically relevant GATA-3 knockdown with subsequent downregulation of Th2 cytokines and reduced eosinophilia. In human precision-cut lung slices (hPCLS), T cells were activated for three days and transfected for two days. GATA3 mRNA levels were assessed using RT-qPCR. Additionally, a multiplex ELISA was performed on the supernatants of hPCLS to evaluate IL-6, TNF-α, IL-1ß, and IL-10 activation.