Determining the Effects of Composition and Microfluidic Fabrication Parameters on Lipid Nanoparticle Structure and Function

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RNA-based gene therapy has potential to cure most diseases but is limited because of challenges in delivering sufficient amounts of RNA to specific tissue or cells. For example, RNA-based gene therapy is used in the leading COVID-19 vaccines, including the one I helped develop, because it is easy to deliver RNA into the muscle. Self-amplifying RNA (saRNA) is a cutting-edge gene delivery platform; by encoding a replicase derived from an alphavirus genome, the RNA is able to replicate upon entry into the cell. The self-replicating properties result in a highly active form of RNA, and saRNA requires ~100-fold lower dose compared to messenger RNA. However, because saRNA is a large (~10,000 nt), anionic molecule, it requires a delivery system to promote cellular uptake. Due to their potency and scalability, lipid nanoparticles (LNPs) are the lead formulation for saRNA. However, current LNPs were optimized for smaller RNA (siRNA, mRNA), and are less suited to saRNA. While cellular activation by RNA is well-characterized, it is less clear how the physical characteristics of LNPs are detected by intracellular mechanisms of cells. We hypothesize that previously undefined nanoparticle characteristics, critical quality attributes (CQAs), can be tuned by the critical processing parameters (CPPs) to increase the functionality and batch-to-batch consistency of LNP formulations containing saRNA. The long-term objective of my research program is to gain a deeper understanding of the mechanisms that govern sensing of saRNA LNPs and to utilize biochemical design and processing parameters to enhance the immunological activity of saRNA formulations. Within the next five years, the short-term objectives are to understand how microfluidic processing parameters affect physical attributes of saRNA LNPs, and how these characteristics are sensed intracellularly: (1)We will use a high throughput Design of Experiments approach to optimize CPPs specifically for saRNA LNPs. (2)We will develop rigorous `fingerprinting' characterization methods and correlation with CPPs to create potent transfection agents for saRNA. (3)We will utilize saRNA LNPs with varying CQAs to understand the mechanisms underpinning intracellular sensing using single cell RNA sequencing and cytokine secretion profiles. These studies will establish the importance of the biological impact of RNA carriers, and not just the RNA itself, within the field of RNA delivery. As an interdisciplinary lab, the 21 HQP included in my overall research program will be trained in chemical engineering, biomaterials, and molecular biology techniques, and I will nurture a research group that promotes equity, diversity and inclusion of HQP at all skill levels and backgrounds. This research program will benefit Canada both by providing multifaceted training for the next generation of gene delivery scientists and developing technologies that directly translate to benefit the emerging and future industry in gene therapy.