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

基于RNA的基因疗法具有治愈大多数疾病的潜力，但由于在向特定组织或细胞输送足够数量的RNA方面存在挑战，因此受到限制。例如，基于RNA的基因疗法被用于领先的COVID-19疫苗，包括我参与开发的疫苗，因为它很容易将RNA传递到肌肉中。自我扩增RNA （saRNA）是一种前沿的基因传递平台；通过编码源自甲病毒基因组的复制酶，RNA能够在进入细胞后进行复制。这种自我复制的特性导致了RNA的高度活性，而saRNA所需的剂量比信使RNA低约100倍。然而，由于saRNA是一个大的（~10,000 nt）阴离子分子，它需要一个传递系统来促进细胞摄取。由于其效力和可扩展性，脂质纳米颗粒（LNPs）是saRNA的主要配方。然而，目前的LNPs针对较小的RNA （siRNA, mRNA）进行了优化，并且不太适合saRNA。虽然RNA对细胞的激活已被充分表征，但LNPs的物理特性如何通过细胞内机制检测尚不清楚。我们假设以前未定义的纳米颗粒特性，关键质量属性（cqa）可以通过关键加工参数（CPPs）进行调整，以增加含有saRNA的LNP配方的功能和批次间一致性。我的研究计划的长期目标是更深入地了解控制saRNA LNPs感知的机制，并利用生化设计和加工参数来增强saRNA配方的免疫活性。在未来五年内，短期目标是了解微流体处理参数如何影响saRNA LNPs的物理属性，以及这些特性如何在细胞内被感知：(1)我们将使用高通量实验设计方法来优化专门针对saRNA LNPs的CPPs。(2)我们将开发严格的“指纹”表征方法和与CPPs的相关性，以创建有效的saRNA转染剂。(3)我们将利用具有不同cqa的saRNA LNPs，利用单细胞RNA测序和细胞因子分泌谱来了解细胞内传感的机制。这些研究将确立RNA载体的生物学影响的重要性，而不仅仅是RNA本身，在RNA递送领域。作为一个跨学科的实验室，我的整体研究计划中的21名HQP将接受化学工程、生物材料和分子生物学技术方面的培训，我将培养一个研究小组，促进所有技能水平和背景的HQP的公平性、多样性和包容性。这项研究计划将为加拿大提供多方面的培训，培养下一代基因传递科学家，并开发直接转化为有利于新兴和未来基因治疗行业的技术。

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.