This document proposes a 5-year $23.967M programme to develop innovative nanoparticle delivery systems for ribonucleic acid (RNA) therapeutics. Our goal is to design programmable nanoparticle delivery systems to overcome the endosomal escape (EE) bottleneck and achieve non-liver organ delivery. We shall accomplish this through advancing the fundamental understanding of the dynamics of nanoparticle intracellular trafficking and its correlation to particle formulation.
RNA therapeutics are believed to have great potential to treat or prevent multiple human diseases ranging from infectious diseases to cancers to genetic disorders. In addition, RNA therapeutics can be personalised, and act on targets that are otherwise “undruggable” by small-molecule or protein drugs.
A primary obstacle to the exploitation of RNA nanomedicine is its delivery. Naked RNAs are negatively charged and are generally too large to enter cells on their own. They are also vulnerable to removal by the immune system and by RNases in tissues. Hence, they require suitable delivery vehicles to reach the cytosol, their target site, for downstream actions. Among various families of delivery vehicles explored, synthetic lipid nanoparticles (LNPs), employed in the recent FDA-approved COVID-19 mRNA-LNP vaccines, are one of the most effective systems.
Though synthetic delivery nanoparticles, including LNPs, can enter cells (primarily via endocytosis), they are mostly trapped inside endosomal compartments and remain separated from the target cytosol. Typically, only a small fraction (2-4%) can successfully deliver their cargoes to the cytosol while the remainder is subsequently expelled or degraded. The ionisable lipid used in Moderna’s mRNA COVID-19 vaccine results in an exceptional EE efficiency (~10%). Much higher gene knock-down or protein expression levels (1000x higher) are required to fully realize the potential of RNA nanomedicines for therapeutic and protein-encoding applications. It is generally agreed that EE is a major rate-limiting step for RNA therapeutics.
Improvement of RNA delivery efficiency has been hindered by poor understanding of the intracellular trafficking and EE process of nanoparticles and how the nanoparticle composition influences these. This is largely due to the interdisciplinary nature of the EE problem and the technical challenges involved. Cell biologists have found that the intracellular trafficking of native cargoes relies on complex networks of proteins and lipids. However, how these intrinsic regulators are tuneable by synthetic LNPs, and the extent to which the LNPs can influence their pathways and eventual fates, are largely unknown. Further, the relevant sizes and time scales of EE are often beyond the spatiotemporal resolution of conventional optical microscopy.
Hence, the aim of this programme is to understand the molecular and cellular mechanisms governing the endosomal trafficking and escape of synthetic nanoparticles in order to achieve efficient and nontoxic cytosolic delivery of RNAs. We also aim to uncover design criteria for programmable nanoparticles that are capable of controllable endosomal escape and intracellular targeting. We shall also design nanoparticle vehicles that can achieve specific non-liver organ targeting.
The programme aims to overcome the poor EE efficiency barrier by the concerted efforts of a cross-disciplinary team. Further, we shall exploit the recent technological advances that the team has made in formulation chemistry, microscopy technique, cellular manipulation, and nanochip for high-throughput screening. Importantly, the study of trafficking dynamics of synthetic nanoparticles, as opposed to native cargoes, is an untapped area. Our effort here is envisioned to bring about exciting discoveries.
This proposal is structured as three interrelated and iterative themes. Theme 1 has two aims: development of (i) a class of programmable LNPs augmented by polymers (P) and peptides (P) (called PP-LNPs) to offer significant improvement of EE efficiency, and (ii) polymersomes with lung-/spleen-targeting ability. Synthetic formulations that are EE-efficient and nontoxic are rare. Viruses, on the other hand, can achieve superior EE efficiency (e.g., 40-70% with enveloped virus) by employing multiple coordinated escape mechanisms. However, viral-based delivery vectors suffer from immunogenicity, high manufacturing cost, etc. Smart PP-LNPs employing multiple coordinated mechanisms due to various EE-effecting agents (lipids, polymers, and peptides) and with endosome-/pathway-responsiveness will be invented. Further, new cationic centers will be exploited, specifically derivatives of azole/azolium (such as imidazole in native histidine) that form transient hydrophobic N-heterocyclic carbenes to increase amphiphilicity leading to improved membrane penetration. Furthermore, polymersomes with tuneable surface charges that we have shown to achieve spleen-/lung-targeting shall be further optimised.
In Theme 2, the molecular details of endosomal trafficking and escape mechanism(s) of PP-LNPs shall be uncovered using advanced super-resolution microscopy and lipid manipulations of live cells, and a nanochip for in vitro reconstitutions of the EE process. The study of the EE process is challenging because it is a transient and rare event that involves nanometre-scale organelles. The fluorescence signals of escaped particles are usually weak. Advanced fast-speed microscopy, such as lattice light sheet microscopy (recently developed by the Tom Kirchhausen lab, part of our team), which allows dynamic capture of events in 3D, shall be applied here to study our programmable synthetic nanoparticles. Further, manipulation of protein expression via the CRISPR/Cas9-based gene editing approach shall be applied to uncover endosomal membrane lipid compositions that enable efficient EE. A nanochip shall be developed for high-throughput screening of biochemical and biophysical parameters of cells and particles that control EE efficiency.
A critical problem in RNA delivery research is that in vitro efficacy does not always translate to in vivo results. In Theme 3, we shall evaluate the in vivo transfection efficacy of optimised PP-LNPs, along with their toxicity and organ selectivity, to ensure that in vitro measurements are relevant to in vivo delivery efficiency. Optimal PP-LNPs shall be assessed with rodent models that avoid the confounding effects from systemic delivery, specifically via (i) vaccine applications and (ii) a scaffold-mediated delivery for neural regeneration. Further, we shall evaluate our particles with human organoids to treat a lung disease. To evaluate formulations that achieve lung-/spleen-targeting with systemic administration, large-scale in vivo parallel screening of PP-LNPs shall be performed using DNA barcode technology with Cre-loxP reporter mice. The organ-targeting formulations shall be evaluated with in vivo models for treating lung cancer and controlling myocardial infarction (via silencing inflammatory monocytes).