Journal
GENE THERAPY
Volume 29, Issue 3-4, Pages 157-170Publisher
SPRINGERNATURE
DOI: 10.1038/s41434-021-00282-6
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Funding
- Science Foundation Ireland [18/FIP/3576, 13/IA/1962]
- CIBERER from Instituto de Salud Carlos III [ER18TRL714]
- Instituto de Salud Carlos III [PI20/00615]
- European Regional Development Funds
- IReL Consortium
- Science Foundation Ireland (SFI) [18/FIP/3576, 13/IA/1962] Funding Source: Science Foundation Ireland (SFI)
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This study demonstrates the potential of the HPAE-EB polymer for CRISPR-Cas9 genome editing, achieving targeted exon excision in the COL7A1 gene through a dual-RNA guide system. The biophysical properties of HPAE-EB were found to be efficient and cytocompatible in HEK293 cells, with a target genomic excision rate of 15-20%, but showed reduced efficiency in RDEB keratinocytes. However, delivery of CRISPR-Cas9 as a ribonucleoprotein complex increased targeted genomic excision to over 40%.
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(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.
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