Periodic Reporting for period 2 - RESPIRNA (REpurposing lung Surfactant Protein B for Inhalation therapy with RNA therapeutics)
Reporting period: 2022-11-01 to 2024-04-30
RNA drugs are an emerging class of therapeutics that address diseases at the genomic and/or transcriptomic level. However, their widespread clinical translation is hampered by many extra-and intracellular barriers. Most importantly, RNAs are negatively charged macromolecules that cannot cross biological membranes, making delivery into diseased target cells challenging. To guide RNA molecules across these barriers, they are generally encapsulated into lipid nanoparticles (LNPs).
The lung is an attractive target organ for RNA delivery. Non-invasive pulmonary instillation of RNA requires lower RNA doses and can substantially reduce the risk of systemic side effects. Of note, pulmonary administration allows direct access to target cells, e.g. epithelial cells and alveolar macrophages. The prevalence of respiratory pathologies is surging and for many an unmet therapeutic need exists, underscoring the need for novel therapies. A variety of interesting therapeutic targets are identified for RNA-mediated therapeutic intervention in lung-related diseases. However, in spite of many advantages of RNA inhalation therapy, to date no innovative LNP-based RNA formulations are available for application in the lung. Moreover, given the transient nature of RNA drugs and the need for repeated administration, questions arise regarding the use of current LNP formulations for chronic treatment, highlighting the need for more biocompatible RNA formulations that merge efficient cellular delivery with acceptable toxicity.
Successful proof-of-concept was obtained that cytosolic delivery of RNA into lung-related target cells can be promoted by exploiting lung surfactant protein B as a naturally occurring RNA delivery enhancer. In the RESPIRNA project, I aim to repurpose this natural surfactant protein for intracellular RNA delivery, (1) through identification of SP-B mimicking peptides that can be chemically synthesized and can equally promote RNA delivery, (2) by exploring SP-B’s cellular mode-of-action towards improved cytosolic delivery of RNA, using state-of-the-art fluorescence microscopy and membrane perturbation assays, (3) by designing multifunctional and multicomponent SP-B (peptide) lipid nanoparticles (LNPs) via microfluidic mixing technology and (4) by applying these nanocarriers for RNA delivery in the lung, using models of eosinophilic asthma and cystic fibrosis.
The lung is an attractive target organ for RNA delivery. Non-invasive pulmonary instillation of RNA requires lower RNA doses and can substantially reduce the risk of systemic side effects. Of note, pulmonary administration allows direct access to target cells, e.g. epithelial cells and alveolar macrophages. The prevalence of respiratory pathologies is surging and for many an unmet therapeutic need exists, underscoring the need for novel therapies. A variety of interesting therapeutic targets are identified for RNA-mediated therapeutic intervention in lung-related diseases. However, in spite of many advantages of RNA inhalation therapy, to date no innovative LNP-based RNA formulations are available for application in the lung. Moreover, given the transient nature of RNA drugs and the need for repeated administration, questions arise regarding the use of current LNP formulations for chronic treatment, highlighting the need for more biocompatible RNA formulations that merge efficient cellular delivery with acceptable toxicity.
Successful proof-of-concept was obtained that cytosolic delivery of RNA into lung-related target cells can be promoted by exploiting lung surfactant protein B as a naturally occurring RNA delivery enhancer. In the RESPIRNA project, I aim to repurpose this natural surfactant protein for intracellular RNA delivery, (1) through identification of SP-B mimicking peptides that can be chemically synthesized and can equally promote RNA delivery, (2) by exploring SP-B’s cellular mode-of-action towards improved cytosolic delivery of RNA, using state-of-the-art fluorescence microscopy and membrane perturbation assays, (3) by designing multifunctional and multicomponent SP-B (peptide) lipid nanoparticles (LNPs) via microfluidic mixing technology and (4) by applying these nanocarriers for RNA delivery in the lung, using models of eosinophilic asthma and cystic fibrosis.
The lung is an attractive target organ for delivery of RNA therapeutics. Unfortunately, currently no key-enabling technologies are available that can assist delivery of RNA-based drugs across pulmonary extra-and intracellular barriers following inhalation therapy. Moreover, existing state-of-the-art lipid nanoparticle (LNP) formulations still suffer from relatively inefficient delivery performance and questions remain regarding their safe applicability following repeated administration in chronic dosing regimens.
To improve intracellular delivery of RNA therapeutics, such as siRNA and mRNA, the RESPIRNA team is developing bio-inspired nanoformulations based on pulmonary surfactant. The latter biomaterial contains the membrane-active and cationic amphiphilic surfactant protein B (SP-B), which was identified as an enhancer of cytosolic RNA delivery via fusion with endosomal membranes. This mechanism of cellular RNA delivery resembles a virus-like mode-of-action. Additionally, an SP-B-derived peptide was discovered which can recapitulate the delivery-promoting activity of the native SP-B. As this peptide can easily be chemically synthesized, this advancement allows to bypass the need for full length SP-B extracted from animal origin.
As an alternative delivery platform, I have successfully repurposed cationic amphiphilic drugs (CADs) as structural and functional components of LNPs. It was demonstrated that CADs can in part or fully replace chemically synthetized ionizable cationic lipids in state-of-the-art LNPs for complexation and subsequent intracellular delivery of mRNA. As CADs are widely used and well-established drugs with distinct pharmacological targets, this repurposed LNP platform (termed CADosomes or CAD-LNPs) can possibly be exploited for drug combination treatment of chronic pathologies.
Finally, in several ongoing projects, my team and I are evaluating distinct LNP formulations optimized for inhalation therapy, e.g. with a stability advantage following aerosolization.
To improve intracellular delivery of RNA therapeutics, such as siRNA and mRNA, the RESPIRNA team is developing bio-inspired nanoformulations based on pulmonary surfactant. The latter biomaterial contains the membrane-active and cationic amphiphilic surfactant protein B (SP-B), which was identified as an enhancer of cytosolic RNA delivery via fusion with endosomal membranes. This mechanism of cellular RNA delivery resembles a virus-like mode-of-action. Additionally, an SP-B-derived peptide was discovered which can recapitulate the delivery-promoting activity of the native SP-B. As this peptide can easily be chemically synthesized, this advancement allows to bypass the need for full length SP-B extracted from animal origin.
As an alternative delivery platform, I have successfully repurposed cationic amphiphilic drugs (CADs) as structural and functional components of LNPs. It was demonstrated that CADs can in part or fully replace chemically synthetized ionizable cationic lipids in state-of-the-art LNPs for complexation and subsequent intracellular delivery of mRNA. As CADs are widely used and well-established drugs with distinct pharmacological targets, this repurposed LNP platform (termed CADosomes or CAD-LNPs) can possibly be exploited for drug combination treatment of chronic pathologies.
Finally, in several ongoing projects, my team and I are evaluating distinct LNP formulations optimized for inhalation therapy, e.g. with a stability advantage following aerosolization.
The recent clinical approval of the first RNAi-based LNP Onpattro® (patisiran) and the SARS-CoV-2 mRNA vaccines (Comirnaty®, Spikevax®) underscore the potential of RNA drugs. Currently, the liver is the organ of choice for systemic siRNA therapies while COVID19 mRNA vaccines are administered intramuscularly. To broaden the therapeutic scope of RNA, a great interest exists in delivering RNA to extrahepatic tissues and applying alternative administration routes.
Although the cationic amphiphilic surfactant protein B (SP-B) has long been known as the key protein in functional mammalian breathing, its possible beneficial role in RNA drug delivery has not been explored in detail. All currently approved LNP formulations consist of four main components, i.e. (i) an ionizable cationic lipid, (ii) a helper (phospho)lipid, (iii) cholesterol and (iv) a PEG-lipid, each having a specific function. The modification of state-of-the-art LNPs with endogenous lung surfactant-derived proteins and/or widely applied cationic amphiphilic drugs (CADs) is expected to provide safe and effective formulations for treatment of chronic pulmonary pathologies and beyond.
Future work will be directed to further optimization of the described (modified) LNP formulations for therapeutic RNA inhalation therapy in relevant models of chronic lung pathologies.
Although the cationic amphiphilic surfactant protein B (SP-B) has long been known as the key protein in functional mammalian breathing, its possible beneficial role in RNA drug delivery has not been explored in detail. All currently approved LNP formulations consist of four main components, i.e. (i) an ionizable cationic lipid, (ii) a helper (phospho)lipid, (iii) cholesterol and (iv) a PEG-lipid, each having a specific function. The modification of state-of-the-art LNPs with endogenous lung surfactant-derived proteins and/or widely applied cationic amphiphilic drugs (CADs) is expected to provide safe and effective formulations for treatment of chronic pulmonary pathologies and beyond.
Future work will be directed to further optimization of the described (modified) LNP formulations for therapeutic RNA inhalation therapy in relevant models of chronic lung pathologies.