Luo Xianjin, Germer Janin, Burghardt Tobias, Grau Melina, Lin Yi, Höhn Miriam, Lächelt Ulrich, Wagner Ernst
Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig-Maximilians-Universität Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.
Center for Nanoscience (CeNS), LMU Munich, 80799 Munich, Germany; Department of Pharmaceutical Sciences, University of Vienna, Josef-Holaubek-Platz 2, 1090 Vienna, Austria.
Eur J Pharm Sci. 2025 Feb 1;205:106983. doi: 10.1016/j.ejps.2024.106983. Epub 2024 Dec 7.
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) protein has been proved as a powerful tool for the treatment of genetic diseases. The Cas9 protein, when combined with single-guide RNA (sgRNA), forms a Cas9/sgRNA ribonucleoprotein (RNP) capable of targeting and editing the genome. However, the limited availability of effective carriers has restricted the broader application of CRISPR/Cas9 RNP. In this study, we evaluated dual pH-responsive amphiphilic xenopeptides (XPs) for delivering CRISPR/Cas9 RNP. These artificial lipo-XPs contain apolar cationizable lipoamino fatty acid (LAF) and polar cationizable oligoaminoethylene acid units such as succinoyl-tetraethylenepentamine (Stp) in various ratios and U-shaped topologies. The carriers were screened for functional Cas9/sgRNA RNP delivery in four different reporter cell lines, including a Duchenne muscular dystrophy (DMD) exon skipping reporter cell model. Significantly enhanced cellular uptake into HeLa cells, effective endosomal disruption in HeLa gal8-mRuby3 cells, and potent genome editing by several Cas9/sgRNA RNP complexes was observed in four different cell lines in the 5 nM sgRNA range. Comparing Cas9/sgRNA RNP complexes with Cas9 mRNA/sgRNA polyplexes in the DMD reporter cell model demonstrated similar splice site editing and high exon skipping of the two different molecular Cas9 modalities. Based on these studies, analogues of two potent U1 LAF-Stp and LAF-Stp structures were deployed, tuning the amphiphilicity of the polar Stp group by replacement with the six oligoamino acids dmGtp, chGtp, dGtp, Htp, Stt, or GEIPA. The most potent LAF-Stp analogues (containing dGtp, chGtp or GEIPA) demonstrated further enhanced gene editing efficiency with EC50 values of 1 nM in the DMD exon skipping reporter cell line. Notably, the EC50 of LAF-dGtp reached 0.51 nM even upon serum incubation. Another carrier (LAF-GEIPA) complexing Cas9/sgRNA RNP and donor DNA, facilitated up to 43 % of homology-directed repair (HDR) in HeLa eGFPd2 cells visualized by the switch from green fluorescent protein (eGFP) to blue fluorescent protein (BFP). This study presents a delivery system tunable for Cas9 RNP complexes or Cas9 RNP/donor DNA polyplexes, offering an effective and easily applicable strategy for gene editing.
成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关（Cas）蛋白已被证明是治疗遗传疾病的有力工具。Cas9蛋白与单向导RNA（sgRNA）结合时，会形成一种能够靶向和编辑基因组的Cas9/sgRNA核糖核蛋白（RNP）。然而，有效载体的可用性有限限制了CRISPR/Cas9 RNP的更广泛应用。在本研究中，我们评估了用于递送CRISPR/Cas9 RNP的双pH响应两亲性异种肽（XPs）。这些人工脂质XPs包含不同比例和U形拓扑结构的非极性可阳离子化脂氨基脂肪酸（LAF）和极性可阳离子化低聚氨基乙烯酸单元，如琥珀酰四亚乙基五胺（Stp）。在四种不同的报告细胞系中筛选了这些载体用于功能性Cas9/sgRNA RNP递送，其中包括杜氏肌营养不良（DMD）外显子跳跃报告细胞模型。在5 nM sgRNA范围内，在四种不同细胞系中观察到HeLa细胞的细胞摄取显著增强、HeLa gal8-mRuby3细胞中的有效内体破坏以及几种Cas9/sgRNA RNP复合物的有效基因组编辑。在DMD报告细胞模型中将Cas9/sgRNA RNP复合物与Cas9 mRNA/sgRNA多聚体进行比较，结果表明两种不同分子形式的Cas9具有相似的剪接位点编辑和高外显子跳跃率。基于这些研究，部署了两种有效的U1 LAF-Stp和LAF-Stp结构的类似物，通过用六种低聚氨基酸dmGtp、chGtp、dGtp、Htp、Stt或GEIPA替代来调节极性Stp基团的两亲性。最有效的LAF-Stp类似物（含有dGtp、chGtp或GEIPA）在DMD外显子跳跃报告细胞系中表现出进一步提高的基因编辑效率，EC50值为1 nM。值得注意的是，即使在血清孵育后，LAF-dGtp的EC50也达到了0.51 nM。另一种将Cas9/sgRNA RNP与供体DNA复合的载体（LAF-GEIPA），在HeLa eGFPd2细胞中促进了高达43%的同源定向修复（HDR），从绿色荧光蛋白（eGFP）转换为蓝色荧光蛋白（BFP）即可观察到。本研究提出了一种可针对Cas9 RNP复合物或Cas9 RNP/供体DNA多聚体进行调节的递送系统，为基因编辑提供了一种有效且易于应用的策略。
