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

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 nucl...

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Autores principales: Xiu, Kemao, Saunders, Laura, Wen, Luan, Ruan, Jinxue, Dong, Ruonan, Song, Jun, Yang, Dongshan, Zhang, Jifeng, Xu, Jie, Chen, Y. Eugene, Ma, Peter X.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9818138/
https://www.ncbi.nlm.nih.gov/pubmed/36611948
http://dx.doi.org/10.3390/cells12010156
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author Xiu, Kemao
Saunders, Laura
Wen, Luan
Ruan, Jinxue
Dong, Ruonan
Song, Jun
Yang, Dongshan
Zhang, Jifeng
Xu, Jie
Chen, Y. Eugene
Ma, Peter X.
author_facet Xiu, Kemao
Saunders, Laura
Wen, Luan
Ruan, Jinxue
Dong, Ruonan
Song, Jun
Yang, Dongshan
Zhang, Jifeng
Xu, Jie
Chen, Y. Eugene
Ma, Peter X.
author_sort Xiu, Kemao
collection PubMed
description 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.
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spelling pubmed-98181382023-01-07 Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing Xiu, Kemao Saunders, Laura Wen, Luan Ruan, Jinxue Dong, Ruonan Song, Jun Yang, Dongshan Zhang, Jifeng Xu, Jie Chen, Y. Eugene Ma, Peter X. Cells Article 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. MDPI 2022-12-30 /pmc/articles/PMC9818138/ /pubmed/36611948 http://dx.doi.org/10.3390/cells12010156 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Xiu, Kemao
Saunders, Laura
Wen, Luan
Ruan, Jinxue
Dong, Ruonan
Song, Jun
Yang, Dongshan
Zhang, Jifeng
Xu, Jie
Chen, Y. Eugene
Ma, Peter X.
Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing
title Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing
title_full Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing
title_fullStr Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing
title_full_unstemmed Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing
title_short Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing
title_sort delivery of crispr/cas9 plasmid dna by hyperbranched polymeric nanoparticles enables efficient gene editing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9818138/
https://www.ncbi.nlm.nih.gov/pubmed/36611948
http://dx.doi.org/10.3390/cells12010156
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