Gene editing of MPS I human fibroblasts by co-delivery of a CRISPR/Cas9 plasmid and a donor oligonucleotide using nanoemulsions as nonviral carriers.
Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by the deficiency of alpha-L-iduronidase (IDUA). This study shows the use of nanoemulsions co-complexed with the plasmid of CRISPR/Cas9 system and a donor oligonucleotide aiming at MPS I gene editing in vitro. Nanoemulsions composed of MCT, DOPE, DOTAP, DSPE-PEG, and water were prepared by high-pressure homogenization. The DNA was complexed by adsorption (NA) or encapsulation (NE) of preformed DNA/DOTAP complexes with nanoemulsions at +4/-1 charge ratio. The incubation in pure DMEM or supplemented with serum showed that the complexation with DNA was stable after 1 h of incubation, but the complexes tended to release the adsorbed DNA after 24 h of incubation, while the encapsulated DNA remained complexed in the oil core of the nanoemulsions even 48 h after incubation with DMEM. The treatment of MPS I patient's fibroblasts homozygous for the p.Trp402∗ mutation led to a significant increase in IDUA activity at 2, 15, and 30 days when compared to MPS I untreated fibroblasts. Flow cytometry and confocal microscopy demonstrated that there was a reduction in the area of lysosomes to values similar to normal, an indicator of correction of the cellular phenotype. These results show that the nanoemulsions co-complexed with the CRISPR/Cas9 system and a donor oligonucleotide could effectively transfect MPS I p.Trp402∗ patient's fibroblasts, as well as enable the production of IDUA, and represent a potential new treatment option for MPS I.
['Schuh|Roselena Silvestri|RS|', 'de Carvalho|Talita Giacomet|TG|', 'Giugliani|Roberto|R|', 'Matte|Ursula|U|', 'Baldo|Guilherme|G|', 'Teixeira|Helder Ferreira|HF|']
D001483:Base Sequence D064113:CRISPR-Cas Systems D002478:Cells, Cultured D004247:DNA D004337:Drug Carriers D004655:Emulsions D005229:Fatty Acids, Monounsaturated D005347:Fibroblasts D000072669:Gene Editing D006801:Humans D007068:Iduronidase D008059:Mucopolysaccharidosis I D053758:Nanoparticles D009841:Oligonucleotides D010714:Phosphatidylethanolamines D010957:Plasmids D011092:Polyethylene Glycols D000644:Quaternary Ammonium Compounds
Gene editing CRISPR/Cas9 Nanoemulsion Mucopolysaccharidosis type I Gene therapy Nonviral vectors