Double pH-responsive xenopeptide carriers containing succinoyl tetraethylene pentamine (Stp) and lipo amino fatty acids (LAFs) were evaluated for CRISPR/Cas9 based genome editing. Different carrier topologies, variation of LAF/Stp ratios and LAF types as Cas9 mRNA/sgRNA polyplexes were screened in three different reporter cell lines using three different genomic targets (, eGFP, exon 23). One U-shaped and three bundle (B2)-shaped lipo-xenopeptides exhibiting remarkable efficiencies were identified. Genome editing potency of top carriers were observed at sub-nanomolar EC concentrations of 0.4 nM sgRNA and 0.1 nM sgRNA for the top U-shape and top B2 carriers, respectively, even after incubation in full (≥ 90%) serum. Polyplexes co-delivering Cas9 mRNA/sgRNA with a single stranded DNA template for homology directed gene editing resulted in up to 38% conversion of eGFP to BFP in reporter cells. Top carriers were formulated as polyplexes or lipid nanoparticles (LNPs) for subsequent administration. Formulations displayed long-term physicochemical and functional stability upon storage at 4 °C. Importantly, intravenous administration of polyplexes or LNPs mediated editing of the dystrophin gene, triggering mRNA exon 23 splicing modulation in dystrophin-expressing cardiac muscle, skeletal muscle and brain tissue.

中文翻译：

---

用于基于 CRISPR/Cas9 的超低剂量基因编辑的脂质异肽复合物

评估了含有琥珀酰四亚乙基五胺 (Stp) 和脂氨基酸脂肪酸 (LAF) 的双 pH 响应型异肽载体，用于基于 CRISPR/Cas9 的基因组编辑。使用三种不同的基因组靶标（eGFP、外显子 23）在三种不同的报告细胞系中筛选不同的载体拓扑、LAF/Stp 比率的变化以及作为 Cas9 mRNA/sgRNA 复合物的 LAF 类型。鉴定出一种 U 形和三束 (B2) 形脂异肽，表现出显着的效率。对于顶级 U 型和顶级 B2 载体，即使在完全 (≥ 90%) 血清中孵育后，在分别为 0.4 nM sgRNA 和 0.1 nM sgRNA 的亚纳摩尔 EC 浓度下观察到顶级载体的基因组编辑效力。 Polyplexs 与单链 DNA 模板共同递送 Cas9 mRNA/sgRNA，用于同源定向基因编辑，导致报告细胞中 eGFP 转化为 BFP 高达 38%。顶级载体被配制为复合物或脂质纳米颗粒（LNP）以供后续施用。 4°C 储存后，制剂表现出长期的理化和功能稳定性。重要的是，静脉注射复合物或 LNP 介导肌营养不良蛋白基因的编辑，触发表达肌营养不良蛋白的心肌、骨骼肌和脑组织中 mRNA 外显子 23 剪接调节。