Periodic Reporting for period 4 - OBSERVE (Overcoming cellular barriers to therapeutic RNA delivery using extracellular vesicles)
Reporting period: 2024-08-01 to 2025-01-31
RNA-based therapeutics, including siRNA, miRNA, mRNA and CRISPR/Cas9 components, have unprecedented therapeutic potential and hold the promise of treating any disease with a genetic component. However, inefficient delivery into diseased cells hinders their clinical progress. Consequently, there is an urgent need for novel, original approaches to overcome the delivery challenges.
Recently, an endogenous RNA transport system has emerged, based on the release and uptake of extracellular vesicles (EVs). EVs are naturally equipped to transfer RNA molecules to other cells in a functional and selective manner. Furthermore, I have recently demonstrated that EVs deliver RNA more efficiently than state-of-the-art synthetic RNA nanocarriers. Thus, EVs hold promise as a new paradigm for RNA delivery. However, the mechanisms underlying EV-mediated RNA transfer are unknown, and reproducible methods for efficient loading of EVs with therapeutic RNA are lacking.
The aim of my proposal is to (1) elucidate the mechanisms underlying EV internalization and processing that lead to the functional delivery of their RNA content and (2) radically improve the loading efficiency of EVs for therapeutic RNA delivery. To realize this, I will:
1) Identify genes and pathways involved in EV-mediated RNA transfer using a novel CRISPR/Cas9-based RNA delivery reporter system.
2) Systematically compare uptake and intracellular trafficking of EVs and synthetic nanocarriers.
3) Design a novel and improved method to load EVs with therapeutic RNA.
4) Demonstrate the effect of EV-mediated delivery of therapeutic RNA in a murine model of myocardial infarction.
The outcome of this research will fundamentally advance our understanding of the cellular processes enabling EV-mediated RNA transfer, which is of crucial importance for the development of EV-based and EV-inspired delivery systems. This research will accelerate clinical translation of an entire new class of therapeutics based on EVs and RNA.
Recently, an endogenous RNA transport system has emerged, based on the release and uptake of extracellular vesicles (EVs). EVs are naturally equipped to transfer RNA molecules to other cells in a functional and selective manner. Furthermore, I have recently demonstrated that EVs deliver RNA more efficiently than state-of-the-art synthetic RNA nanocarriers. Thus, EVs hold promise as a new paradigm for RNA delivery. However, the mechanisms underlying EV-mediated RNA transfer are unknown, and reproducible methods for efficient loading of EVs with therapeutic RNA are lacking.
The aim of my proposal is to (1) elucidate the mechanisms underlying EV internalization and processing that lead to the functional delivery of their RNA content and (2) radically improve the loading efficiency of EVs for therapeutic RNA delivery. To realize this, I will:
1) Identify genes and pathways involved in EV-mediated RNA transfer using a novel CRISPR/Cas9-based RNA delivery reporter system.
2) Systematically compare uptake and intracellular trafficking of EVs and synthetic nanocarriers.
3) Design a novel and improved method to load EVs with therapeutic RNA.
4) Demonstrate the effect of EV-mediated delivery of therapeutic RNA in a murine model of myocardial infarction.
The outcome of this research will fundamentally advance our understanding of the cellular processes enabling EV-mediated RNA transfer, which is of crucial importance for the development of EV-based and EV-inspired delivery systems. This research will accelerate clinical translation of an entire new class of therapeutics based on EVs and RNA.
Regarding objective 1, after optimization of assay parameters, we have performed a small scale RNAi screen. This has allowed us to identify novel genes and pathways involved in RNAi-mediated RNA transfer. We have followed up on one lead target. We have been able to validate the contribution of this lead target, and discover its binding partner on EVs. A manuscript describing these findings has been published in Journal of Biological Chemistry.
Regarding objective 2, we have optimized methods for labelling EV subpopulations and their RNA cargo and are currently analyzing their uptake efficiency and colocalization with endosomal markers as a function of time. In addition, we have set up a novel method to evaluate cargo release efficiencies for different EV subpopulations. We have discovered that EV subpopulations differ in their intracellular trafficking and cargo release efficiencies, which has implications for the use of EVs for drug delivery.
Regarding objective 3, we have started to optimize conditions for fusion or coating of synthetic nanoparticles with EVs, and are performing in-depth investigations to characterize these hybrid structures. In addition, we have set up a novel method to incorporate membrane protein extracts from cells or EVs into lipid nanoparticles, and load these hybrid structures with mRNA. We have shown that these hybrids are internalized and deliver mRNA more than LNPs alone. A manuscript describing these findings has been submitted (figure 1).
Regarding objective 4, we have set up the methodology for evaluating LNP/hybrid biodistribution and mRNA delivery efficiency in mouse model of myocardial infarction. We have performed a biodistribution study of LNPs vs hybrids in this mouse model of MI, which showed some, but only sublte, differences in biodistribution. We are still optimizing our hybrid structures before therapeutic in vivo evaluation.
Regarding objective 2, we have optimized methods for labelling EV subpopulations and their RNA cargo and are currently analyzing their uptake efficiency and colocalization with endosomal markers as a function of time. In addition, we have set up a novel method to evaluate cargo release efficiencies for different EV subpopulations. We have discovered that EV subpopulations differ in their intracellular trafficking and cargo release efficiencies, which has implications for the use of EVs for drug delivery.
Regarding objective 3, we have started to optimize conditions for fusion or coating of synthetic nanoparticles with EVs, and are performing in-depth investigations to characterize these hybrid structures. In addition, we have set up a novel method to incorporate membrane protein extracts from cells or EVs into lipid nanoparticles, and load these hybrid structures with mRNA. We have shown that these hybrids are internalized and deliver mRNA more than LNPs alone. A manuscript describing these findings has been submitted (figure 1).
Regarding objective 4, we have set up the methodology for evaluating LNP/hybrid biodistribution and mRNA delivery efficiency in mouse model of myocardial infarction. We have performed a biodistribution study of LNPs vs hybrids in this mouse model of MI, which showed some, but only sublte, differences in biodistribution. We are still optimizing our hybrid structures before therapeutic in vivo evaluation.
- We have identified ITGB1 as a receptor for EVs which allows EV entry and functional RNA transfer. We identified a4 as its heterodimer and discovered the molecular mechanism through which it interacts with EVs. A manuscript describing these findings has been published in Journal of Biological Chemistry.
- We have found evidence of differential trafficking and cargo release efficiences for different EV subpopulations. We are continuing to study the underlying mechanisms, and see how we can exploit these insights for better drug delivery.
- We have discovered that incorporation of cellular membrane protein extracts into lipid nanoparticles enhances their cellular uptake and mRNA delivery efficiency. A manuscript describing these findings has been published in Journal of Controlled Release. We can now also use this methodology for scalable preparation of EV mimetics.
- We have found evidence of differential trafficking and cargo release efficiences for different EV subpopulations. We are continuing to study the underlying mechanisms, and see how we can exploit these insights for better drug delivery.
- We have discovered that incorporation of cellular membrane protein extracts into lipid nanoparticles enhances their cellular uptake and mRNA delivery efficiency. A manuscript describing these findings has been published in Journal of Controlled Release. We can now also use this methodology for scalable preparation of EV mimetics.