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

Authors :
Kemao Xiu
Laura Saunders
Luan Wen
Jinxue Ruan
Ruonan Dong
Jun Song
Dongshan Yang
Jifeng Zhang
Jie Xu
Y. Eugene Chen
Peter X. Ma
Source :
Cells, Vol 12, Iss 1, p 156 (2022)
Publication Year :
2022
Publisher :
MDPI AG, 2022.

Abstract

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.

Details

Language :
English
ISSN :
20734409
Volume :
12
Issue :
1
Database :
Directory of Open Access Journals
Journal :
Cells
Publication Type :
Academic Journal
Accession number :
edsdoj.93602edda32c46029d7ed99e5661f88a
Document Type :
article
Full Text :
https://doi.org/10.3390/cells12010156