Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system

Abstract

Recent 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(β-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.

A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide

“…Lastly, we have shown that PMBN enhanced electrotransformation can be used to optimise the delivery of DNA–protein complexes (e.g. for TIS library construction) and we expect that this could also potentially be extended to developing a delivery system for ribonucleoprotein (RNP)-mediated CRISPR genome editing 29 in bacterial cells for efficient targeted mutagenesis.…”

A method for increasing electroporation competency of Gram-negative clinical isolates by Polymyxin B nonapeptide

“…Lastly, we have shown that PMBN enhanced electrotransformation can be used to optimise the delivery of DNA-protein complexes (e.g. for TIS library construction) and we expect that this could also potentially be extended to developing a delivery system for ribonucleoprotein (RNP)-mediated CRISPR genome editing 29 in bacterial cells for e cient targeted mutagenesis.…”

Gene Therapy: The Next-Generation Therapeutics and Their Delivery Approaches for Neurological Disorders

“…Other advantages of the non-viral vectors would be the ease of production, thereby, cost-effectiveness, and their ability to be engineered ( Ramamoorth and Narvekar, 2015 ). On top of that, non-viral vectors can transfer bigger sizes of nucleic acid residues and/or proteins ( Morrell et al, 2008 ; Chang et al, 2017 ; Lee et al, 2018 ; Park et al, 2019 ; Zhang et al, 2021 ; Jubair et al, 2021 ; O'Keeffe Ahern et al, 2022 ). Considering the current progress in gene editing technology, transferring larger biomolecule complexes is desired.…”