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.
Highlights
Gene therapy has long been heralded as a new breakthrough in modern molecular medicine as it enables the treatment of disorders at the genetic level [1,2,3]
highly branched PAE (HPAE)-EB polymer efficiently complexes and encapsulates clustered regulatory interspaced short palindromic repeats (CRISPR)-C7 plasmid Inefficient complexation of nucleic acids leads to suboptimal delivery to target cells
At higher w/w ratios between 15:1 and 30:1, a marked decrease in DNA signal intensity can be attributed to the strong complexation by HPAEEB shielding the CRISPR-C7 plasmid and reducing its accessibility for the DNA stain to intercalate with the plasmid
Summary
Gene therapy has long been heralded as a new breakthrough in modern molecular medicine as it enables the treatment of disorders at the genetic level [1,2,3]. Delivery strategies of gene therapy relying on viral vectors, whilst efficient, raise safety concerns regarding immunogenicity and insertional mutagenesis [4,5,6,7]. Nonviral vectors such as cationic polymers offer an attractive alternative to viruses given their facile synthesis, versatility and improved safety profile [8,9,10]. Breakthrough research by Zhou et al has successfully developed novel highly branched PAE (HPAE) polymers for gene delivery that have outperformed their linear counterparts by orders of magnitude in delivering large DNA constructs into cells [2, 3, 12,13,14, 24,25,26,27,28,29,30,31]
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