Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing

被引:7
作者
Xiu, Kemao [1 ]
Saunders, Laura [2 ]
Wen, Luan [3 ]
Ruan, Jinxue [3 ]
Dong, Ruonan [1 ]
Song, Jun [3 ]
Yang, Dongshan [3 ]
Zhang, Jifeng [3 ]
Xu, Jie [3 ]
Chen, Y. Eugene [3 ]
Ma, Peter X. X. [1 ,2 ,4 ,5 ]
机构
[1] Univ Michigan, Dept Biol & Mat Sci, Ann Arbor, MI 48109 USA
[2] Univ Michigan, Macromol Sci & Engn Ctr, Ann Arbor, MI 48109 USA
[3] Univ Michigan, Ctr Adv Models & Translat Sci & Therapeut, Ann Arbor, MI 48109 USA
[4] Univ Michigan, Dept Biomed Engn, Ann Arbor, MI 48109 USA
[5] Univ Michigan, Dept Mat Sci & Engn, Ann Arbor, MI 48109 USA
基金
美国国家卫生研究院;
关键词
nanoparticle; gene delivery; pDNA; CRISPR; Cas9; PEI; polyplex; gene editing; THERAPY; RNA; PROSPECTS;
D O I
10.3390/cells12010156
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Gene editing nucleases such as CRISPR/Cas9 have enabled efficient and precise gene editing in vitro and hold promise of eventually achieving in vivo gene editing based therapy. However, a major challenge for their use is the lack of a safe and effective virus-free system to deliver gene editing nuclease elements. Polymers are a promising class of delivery vehicle due to their higher safety compared to currently used viral vectors, but polymers suffer from lower transfection efficiency. Polymeric vectors have been used for small nucleotide delivery but have yet to be used successfully with plasmid DNA (pDNA), which is often several hundred times larger than small nucleotides, presenting an engineering challenge. To address this, we extended our previously reported hyperbranched polymer (HP) delivery system for pDNA delivery by synthesizing several variants of HPs: HP-800, HP-1.8K, HP-10K, HP-25K. We demonstrate that all HPs have low toxicity in various cultured cells, with HP-25K being the most efficient at packaging and delivering pDNA. Importantly, HP-25K mediated delivery of CRISPR/Cas9 pDNA resulted in higher gene-editing rates than all other HPs and Lipofectamine at several clinically significant loci in different cell types. Consistently, HP-25K also led to more robust base editing when delivering the CRISPR base editor "BE4-max" pDNA to cells compared with Lipofectamine. The present work demonstrates that HP nanoparticles represent a promising class of vehicle for the non-viral delivery of pDNA towards the clinical application of gene-editing therapy.
引用
收藏
页数:14
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