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

确定成分和微流体制造参数对脂质纳米颗粒结构和功能的影响

• 批准号: RGPIN-2021-02931
• 负责人: Blakney, Anna
• 金额: $ 2.4万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760912
• 关键词: Determining; Effects; Composition; Microfluidic; Fabrication

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.

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递送足够量的RNA方面存在挑战而受到限制。例如，基于RNA的基因疗法被用于领先的COVID-19疫苗，包括我帮助开发的疫苗，因为它很容易将RNA输送到肌肉中。自扩增RNA（saRNA）是一种尖端的基因递送平台;通过编码来自甲病毒基因组的复制酶，RNA能够在进入细胞后复制。自我复制的特性导致了RNA的高度活性形式，与信使RNA相比，saRNA需要低约100倍的剂量。然而，由于saRNA是一种大的（~ 10，000 nt）阴离子分子，它需要一个递送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（LNP）是saRNA的主要制剂。然而，目前的LNP针对较小的RNA（siRNA，mRNA）进行了优化，并且不太适合saRNA。虽然RNA对细胞的激活作用已得到充分表征，但LNP的物理特性如何通过细胞内机制检测尚不清楚。我们假设之前未定义的纳米颗粒特征、关键质量属性（CQA）可以通过关键加工参数（CPP）进行调整，以提高含有saRNA的LNP制剂的功能性和批间一致性。我的研究计划的长期目标是更深入地了解控制saRNA LNP传感的机制，并利用生化设计和加工参数来增强saRNA制剂的免疫活性。在接下来的五年内，短期目标是了解微流体处理参数如何影响saRNA LNP的物理属性，以及这些特征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 LNP的CPP。(2)We将开发严格的“指纹”表征方法和与CPP的相关性，以创建用于saRNA的有效转染剂。(3)We将利用具有不同CQA的saRNA LNP来理解支持使用单细胞RNA测序和细胞因子分泌谱的细胞内传感的机制。这些研究将确定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，我的整个研究计划中包括的21名HQP将接受化学工程，生物材料和分子生物学技术的培训，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。这项研究计划将通过为下一代基因传递科学家提供多方面的培训，并开发直接转化为有利于基因治疗新兴和未来产业的技术，使加拿大受益。