转染 CRISPR/Cas9 质粒 DNA 的多肽树枝状聚合物:优化与机制

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Susanna Zamolo, Elena Zakharova, Lise Boursinhac, Florian Hollfelder, Tamis Darbre and Jean-Louis Reymond
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引用次数: 0

摘要

CRISPR/Cas9 基因编辑技术提供了很好的治疗机会,但需要将大型质粒 DNA(pDNA)转入细胞,而转染试剂比病毒载体更适合这项任务。在这里,我们对 Z22 进行了结构-活性关系研究,Z22 是一种 D-对映体、含精氨酸、脂化肽树枝状聚合物,开发用于 CRISPR/Cas9 质粒共表达 GFP 的 pDNA 转染。虽然所有测试过的树枝状聚合物类似物都能与 pDNA 紧密结合,并将货物内化到细胞中,但 D 手性被证明是成功转染的关键,因为它能避免树枝状聚合物结构被蛋白酶降解,而这种降解是内质体逃逸和可能穿过核膜所必需的。此外,Z22 核心的一个半胱氨酸残基被证明是非必需的,因此被移除以产生活性更强的类似物 Z34,该类似物已被证明能够实现 CRISPR/Cas9 介导的功能性基因编辑。多肽树枝状聚合物很容易通过固相多肽合成获得,而且其序列可以针对特定应用进行微调,因此是有吸引力进一步开发的试剂。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Peptide dendrimers transfecting CRISPR/Cas9 plasmid DNA: optimization and mechanism†

Peptide dendrimers transfecting CRISPR/Cas9 plasmid DNA: optimization and mechanism†

Gene editing by CRISPR/Cas9 offers great therapeutic opportunities but requires delivering large plasmid DNA (pDNA) into cells, a task for which transfection reagents are better suited than viral vectors. Here we performed a structure–activity relationship study of Z22, a D-enantiomeric, arginine containing, lipidated peptide dendrimer developed for pDNA transfection of a CRISPR/Cas9 plasmid co-expressing GFP. While all dendrimer analogs tested bound pDNA strongly and internalized their cargo into cells, D-chirality proved essential for transfection by avoiding proteolysis of the dendrimer structure required for endosome escape and possibly crossing of the nuclear envelope. Furthermore, a cysteine residue at the core of Z22 proved non-essential and was removed to yield the more active analog Z34. This dendrimer shows >83% GFP transfection efficiency in HEK cells with no detrimental effect on cell viability and promotes functional CRISPR/Cas9 mediated gene editing. It is accessible by solid-phase peptide synthesis and therefore attractive for further development.

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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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