Engineering Next-Generation Nanoformulations for Bioresponsive CRISPR/Cas9 Delivery

设计用于生物响应性 CRISPR/Cas9 传递的下一代纳米制剂

• 批准号: 571574-2021
• 负责人: Chen, GuojunG
• 金额: $ 3.28万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Alliance Grants
• 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=760847
• 关键词: Engineering; Next; Generation; Nanoformulations; Bioresponsive

The CRISPR/Cas9 system has emerged as the most exciting genome editing tool for widespread applications in fundamental biological research, bioengineering, agriculture, and healthcare. However, the greatest challenge to its deployment for genetic engineering is the lack of safe, effective, and selective delivery of CRISPR/Cas9 elements.Most CRISPR/Cas9 delivery systems still rely on viral approaches. Although viral vectors offer good delivery performance, they are limited by restricted packaging capacities and safety concerns. Non-viral delivery via nanoparticles (NPs) has the potential to overcome these limitations. While recent efforts have been focused on designing desired CRISPR/Cas9 non-viral vectors, there are still challenges lying ahead. 1) Efficiency: The efficiency of the existing non-viral NPs for CRISPR/Cas9 remains modest. These NPs are formed mainly by self-assembly via weak interactions with poor stability, which leads to premature release of CRISPR/Cas9, thus compromising delivery efficiency. 2) Precision: Current non-viral delivery approaches also lack the ability to target specific cells/tissues for precise genome editing. Therefore, it is extremely valuable to develop a non-viral delivery system that can simultaneously achieve high efficiency and precision in CRISPR/Cas9-mediated genome editing.Calcium Carbonate (CaCO3) NPs have received wide attention in drug delivery, due to their excellent biocompatibility, simple fabrication process, and most importantly, superior stability. Advances in nanotechnology have led to the development of polymer/CaCO3 (pCaCO3) hybrid NPs with controllable physicochemical properties. These advantages place pCaCO3 NPs as an ideal candidate as a drug nanocarrier for CRISPR/Cas9, offering solutions to achieve efficient and precise genome editing, which, however, has not been previously investigated. Hence, the objective of this proposal is to engineer pCaCO3 NP systems for bioresponsive CRISPR/Cas9 delivery to achieve efficient and precise genome editing. We have three specific aims.Aim 1: Design and optimize pCaCO3 NPs for CRISPR/Cas9 ribonucleoprotein (RNP) delivery. Aim 2: Investigate effects of surface physicochemical properties of RNP@pCaCO3 NPs on selective cell/tissue targeting. Aim 3: Engineer pCaCO3 NPs for co-delivering RNP and donor DNA template for gene correction. This research will establish a new generation of non-viral delivery systems for precise and efficient genome editing. The resultant technology will significantly broaden the application of genome editing tools in diverse settings across fundamental science to applied research. New discoveries in the effects of chemical selection and materials design on nano-bio interactions will further contribute to the development of advanced drug delivery systems and biomaterials.

CRISPR/Cas9系统已成为最令人兴奋的基因组编辑工具，广泛应用于基础生物研究，生物工程，农业和医疗保健。然而，将其用于基因工程的最大挑战是缺乏安全、有效和选择性的CRISPR/Cas9元件递送。大多数CRISPR/Cas9递送系统仍然依赖于病毒方法。尽管病毒载体提供良好的递送性能，但它们受到有限的包装能力和安全性问题的限制。通过纳米颗粒（NPs）的非病毒递送具有克服这些限制的潜力。虽然最近的努力集中在设计所需的CRISPR/Cas9非病毒载体上，但未来仍存在挑战。1)效率：现有的CRISPR/Cas9非病毒纳米颗粒的效率仍然很低。这些纳米颗粒主要通过稳定性差的弱相互作用自组装形成，导致CRISPR/Cas9过早释放，从而影响递送效率。2)精确性：目前的非病毒递送方法也缺乏靶向特定细胞/组织进行精确基因组编辑的能力。因此，开发一种能够同时实现CRISPR/Cas9介导的基因组编辑的高效和精确的非病毒递送系统是非常有价值的。碳酸钙（CaCO3）纳米粒由于其优异的生物相容性、简单的制备工艺以及最重要的上级稳定性而在药物递送方面受到广泛关注。纳米技术的进步导致了具有可控物理化学性质的聚合物/CaCO3（pCaCO3）杂化纳米颗粒的发展。这些优势使pCaCO3 NPs成为CRISPR/Cas9药物纳米载体的理想候选者，为实现高效和精确的基因组编辑提供了解决方案，然而，这一点以前尚未研究过。因此，该提案的目的是设计用于生物响应性CRISPR/Cas9递送的pCaCO3 NP系统，以实现高效和精确的基因组编辑。我们有三个具体目标：目标1：设计和优化用于CRISPR/Cas9核糖核蛋白（RNP）递送的pCaCO3 NPs。目的2：研究RNP@pCaCO3纳米粒的表面物理化学性质对选择性细胞/组织靶向的影响。目的3：工程化pCaCO3 NPs用于共递送RNP和供体DNA模板以用于基因校正。这项研究将建立新一代的非病毒递送系统，用于精确和高效的基因组编辑。由此产生的技术将大大拓宽基因组编辑工具在基础科学到应用研究的各种环境中的应用。化学选择和材料设计对纳米生物相互作用影响的新发现将进一步促进先进药物输送系统和生物材料的发展。