Genodermatoses are genetic skin disorders with currently no curative treatment. The fast-pacedadvancement of gene therapy tools offers a solution for the treatment and cure ofgenodermatoses. Autosomal Recessive Congenital Ichthyosis (ARCI) is a genetic skin disorderthat greatly affects the life-quality of the patient. Here, we hypothesized that the gene editing toolCRISPR-Cas9 can be delivered by lipid nanoparticles into primary human keratinocytes and skintissue, and genetically edit the epidermis cells. Initially, we screened different formulations ofLNPs 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 highergene editing efficacy for RNP (14%) and mRNA (15%) approaches. After selecting oneformulation in a 2D in vivo approach, we moved to 3D systems: reconstructed skin models andex vivo excised skin. Also, to improve the penetration of the LNPs into the epidermis, we used400μm solid microneedles that can create micro-sized openings in the skin and facilitate thepermeation of particles. Skin models showed gene editing efficacy of 8-15% for both RNP- andmRNA-loaded LNPs. Similarly, excised human skin indicates gene editing of 9% for RNPloadedand 7% for mRNA-loaded LNPs. To further explore the capacity of CRISPR-loadedLNPs, we tested LNP formulations varying in ionizable lipids. LNPs A-J formulated withdifferent ionizable lipids indicates a significant increase in gene editing efficacy, especially forLNP E (37%) and LNP H (72%), but impaired cell viability (50%). We also tested the additionof saponins as endosomal escape enhancer, but there were no significant changes in gene editingefficacy. 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 alower cell viability. In conclusion, results suggest LNPs loaded with CRISPR-Cas9 componentscan be delivered in primary human keratinocytes and skin tissue and induce in situ genomeediting.
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