Defining the molecular interactions within nanoparticles that enable delivery of long nucleic acids

项目摘要 Cas9 mRNA和靶向sgRNA的共递送是实现CRISPR/Cas基因编辑的一种有前途的策略。 提高了安全性。虽然在短RNA（siRNA）的递送方面已经取得了很大的进展， miRNA），对于较长的RNA货物（mRNA， sgRNA）。我们最近克服了这一递送挑战，报道了第一次成功的NP共递送mRNA 和sgRNA。在这项拨款提案中，我们的目标是从根本上了解为什么两性离子氨基脂质（ZAL） 纳米颗粒（ZNP）独特地适合于并且特别有效地用于递送长RNA。我们假设 NP内两性离子和阳离子基团的分子平衡对于长核酸递送是必需的。 酸，特别是载体分子和RNA之间的界面处的非共价键合力。这 一个假设得到了计算模型的支持，表明磷脂的作用是溶解RNA 在多组分NP内的水性袋内。事实上，我们通过实验发现， 两性离子脂质和阳离子脂质的化学和结构作用转化为单一脂质化合物（ZAL）， 改善长RNA的递送。在这个建议中，我们将开发（并从根本上理解）改进的 用于共递送Cas9 mRNA和靶向sgRNA以实现安全有效的CRISPR/Cas的递送载体 体内基因编辑完成拟议的研究将：（1）确定新ZAL的功能作用 用于mRNA和sgRNA的共递送的头部基团、接头和疏水结构域尾部;（2）鉴定 有效的长RNA（mRNA，sgRNA）脂质载体的理化性质，其与脂质载体的生物学特性相关。 胞内递送机制;以及（3）确定ZALs的化学结构如何介导细胞和 通过利用基因工程Lox-Stop-Lox tdTomato进行体内组织特异性CRISPR/Cas基因编辑 一种可以揭示任何器官或细胞中编辑的鼠标。累积起来，这将为CRISPR/Cas- 通过开发有效、安全和临床可转化的纳米颗粒， 载波定义特定的相互作用是一个关键的目标，这将大大改善长RNA的递送， 现有和未来的航空公司。这种更广泛的影响可能会大大加快临床发展， mRNA和CRISPR/Cas治疗。

Project Summary Co-delivery of Cas9 mRNA and targeted sgRNA is a promising strategy to achieve CRISPR/Cas gene editing in vivo with improved safety. Although great advances have been made in the delivery of short RNAs (siRNA, miRNA), the ideal chemical and formulation composition is largely unknown for longer RNA cargo (mRNA, sgRNA). We recently overcame this delivery challenge in reporting the first successful NP co-delivery of mRNA and sgRNA in vivo. In this grant proposal, we aim to fundamentally understand why zwitterionic amino lipid (ZAL) nanoparticles (ZNPs) are uniquely suitable and particularly efficacious for delivery of long RNAs. We hypothesize that the molecular balance of zwitterionic and cationic groups within NPs is essential for delivery of long nucleic acids, especially the noncovalent bonding forces at the interface between carrier molecules and RNAs. This hypothesis is supported by computational modeling that indicates the role of phospholipids is to solubilize RNAs inside of aqueous pockets within multi-component NPs. Indeed, we found experimentally that combining the chemical and structural roles of zwitterionic lipids and cationic lipids into a single lipid compound (ZAL) greatly improved delivery of long RNAs. In this proposal, we will develop (and fundamentally understand) improved delivery carriers for co-delivery of Cas9 mRNA and targeted sgRNA to enable safe and efficacious CRISPR/Cas gene editing in vivo. Completion of the proposed studies will: (1) Determine the functional roles of novel ZAL headgroups, linkers, and hydrophobic domain tails for co-delivery of mRNA and sgRNA; (2) Identify the physiochemical properties of efficacious long RNA (mRNA, sgRNA) lipid carriers that are correlated to the mechanism of intracellular delivery; and (3) Determine how the chemical structure of ZALs mediate cell and tissue specific CRISPR/Cas gene editing in vivo by utilizing a genetically engineered Lox-Stop-Lox tdTomato mouse that can reveal editing in any organ or cell. Cumulatively, this will open new avenues for CRISPR/Cas- based correction of genetic diseases by developing efficacious, safe, and clinically translatable nanoparticle carriers. Defining the specific interactions is a critical goal that would greatly improve delivery of long RNAs by numerous existing and future carriers. This broader impact may greatly accelerate the clinical development of mRNA and CRISPR/Cas therapeutics.