Ahern, Jonathan O'KeeffeLara-Saez, IreneZhou, DezhongMurillas Angoiti, RodolfoBonafont Aragó, JoséMencía Rodríguez, ÁngelesGarcía Díez, MartaManzanares, DarioLynch, JenniferFoley, RuthXu, QuianSigen, A.Larcher Laguzzi, FernandoWang, Wenxin2024-02-052024-02-052022-04-01O’Keeffe Ahern, J., Lara-Sáez, I., Zhou, D. et al. (2022) Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system. Gene Ther 29, 157–170. https://doi.org/10.1038/s41434-021-00282-60969-7128https://hdl.handle.net/10016/39853Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(beta-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15-20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR-Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.14eng© The Author(s) 2021Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Atribución 3.0 EspañaBranched poly (Beta-Amino Esters)In-vivoTransfectionEfficiencyTherapyEnhanceDNAExpressionChallengesProspectsresearch articleBiología y BiomedicinaMedicinahttps://doi.org/10.1038/s41434-021-00282-6open access1572-3170Gene Therapy29AR/0000034115