Journal of Nanomedicine & Nanotechnology

In situ gene therapy via non-viral delivery of CRISPR-Cas9 to the skin aiming for recovery of autosomal recessive congenital ichthyosis (ARCI)

Joint Meet on 29^th International Conference on Nanomedicine and Nanomaterials & 24^th World Nanotechnology Congress

April 26, 2021 | Webinar

Qurrat Ul Ain*, Danny Liu, Dominik Witzigmann, Jayesh Kulkarni, Ariel Huyhn, Partho Adhikary, Russ Algar, Pieter Cullis, Sarah Hedtrich

Faculty of Pharmaceutical Sciences, University of British Columbia, 2405 Wesbrook Mall, V6T1Z3, Vancouver, BC, Canada
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada
NanoMedicines Innovation Network (NMIN), University of British Columbia, Vancouver, BC, Canada*

Scientific Tracks Abstracts: J Nanomed Nanotechnol

Abstract:

Autosomal recessive congenital ichthyosis (ARCI) is a rare, but severe keratinisation disorder characterized with regions of dry scaled skin, impaired skin barrier function, higher transepidermal water loss and a meaningful susceptibility to infection. Gene editing tools like CRISPR-Cas9 are the ideal for correcting rare monogenic skin diseases like ARCI. We aimed to efficiently deliver CRISPR-Cas9 components (Cas9 protein (RNP) or Cas9-mRNA) to primary human keratinocytes (KCs) and skin stem cells (HPEKPs) using lipid-based nanoparticles (LNPs) for homology directed repair of TGM1 gene. Fluorescently labelled LNPs composed with different helper lipids including DSPC, DOPC, DOPE, DSPG and ES, were optimized systematically as high-performance gene delivery vectors via cellomics quantitative cell analysis. Concurrently, ApoE addition and 0.5%, 1.5% & 5% concentrations of polyethylene glycol (PEG) in LNPs were also assessed. Among the 5 helper lipids; DOPE showed the highest levels of cellular uptake, the inclusion of ApoE exhibited up to a 12.7-fold increase in LNPs uptake. Increasing PEG up to 5% resulted in a decrease in cellular uptake likely due to a physical steric barrier effect. Furthermore, 80-90% of CRISPR-RNAs were encapsulated with LNPs whereas RNPs did not show any significant encapsulation. Through RNPs/LNPs formulations, 12-17% cutting of genomic DNA was achieved while with CRISPR-RNAs it increased up to 30% with no significant cytotoxicity. To observe the effect of addition of permanently charged cationic lipids on LNPs, DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), was added to the LNPs composed of DOPE. Increased cutting efficiencies were seen with the addition of ≤ 10% DOTMA, which however, was also associated with cytotoxicity. Further increase of DOTMA up to 30% and 50% did not further improve the cutting efficiencies, due to strong cytotoxic effects. Conclusively, for efficient delivery of CRISPR-Cas9 components to skin cells comprehensive screening of individual components of nanoparticles libraries is required.