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de Andrade Bolsoni, Juliana

University of British Columbia

Genodermatoses are genetic skin disorders with currently no curative treatment. The fast-paced advancement of gene therapy tools offers a solution for the treatment and cure of genodermatoses. Autosomal Recessive Congenital Ichthyosis (ARCI) is a genetic skin disorder that greatly affects the life-quality of the patient. Here, we hypothesized that the gene editing tool CRISPR-Cas9 can be delivered by lipid nanoparticles into primary human keratinocytes and skin tissue, and genetically edit the epidermis cells. Initially, we screened different formulations of LNPs for physical (size, PDI, zeta potential) and biological (cell viability, gene editing efficacy) characteristics. The first trials suggested DOPE LNPs shows less cytotoxicity (>60%) and higher gene editing efficacy for RNP (14%) and mRNA (15%) approaches. After selecting one formulation in a 2D in vivo approach, we moved to 3D systems: reconstructed skin models and ex vivo excised skin. Also, to improve the penetration of the LNPs into the epidermis, we used 400μm solid microneedles that can create micro-sized openings in the skin and facilitate the permeation of particles. Skin models showed gene editing efficacy of 8-15% for both RNP- and mRNA-loaded LNPs. Similarly, excised human skin indicates gene editing of 9% for RNPloaded and 7% for mRNA-loaded LNPs. To further explore the capacity of CRISPR-loaded LNPs, we tested LNP formulations varying in ionizable lipids. LNPs A-J formulated with different ionizable lipids indicates a significant increase in gene editing efficacy, especially for LNP E (37%) and LNP H (72%), but impaired cell viability (<50%). We also tested the addition of saponins as endosomal escape enhancer, but there were no significant changes in gene editing efficacy. Finally, we tested the use of modified LNPs by adding the cell penetrating peptide Tat. The Tat-LNPs showed an increase in GFP expression when compared to DOPE LNPs, but a lower cell viability. In conclusion, results suggest LNPs loaded with CRISPR-Cas9 components can be delivered in primary human keratinocytes and skin tissue and induce in situ genome editing.

Text

2024-03-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/84132

Pharmaceutical Sciences

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/