Delivery and assessment of a CRISPR/nCas9-based genome editing system on in vitro models of mucopolysaccharidoses IVA assisted by magnetite-based nanoparticles

Abstract

Mucopolysaccharidosis IV A (MPS IVA) is a lysosomal disorder caused by mutations in the GALNS gene. Consequently, the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate accumulate in the lysosomal lumen. Although enzyme replacement therapy has shown essential advantages for the patients, several challenges remain to overcome, such as the limited impact on the bone lesion and recovery of oxidative profile. Recently, we validated a CRISPR/nCas9-based gene therapy with promising results in an in vitro MPS IVA model. In this study, we have expanded the use of this CRISPR/nCas9 system to several MPS IVA fibroblasts carrying different GALNS mutations. Considering the latent need to develop more safety vectors for gene therapy, we co-delivered the CRISPR/nCas9 system with a novel non-viral vector based on magnetoliposomes (MLPs). We found that the CRISPR/nCas9 treatment led to an increase in enzyme activity between 5 and 88% of wild-type levels, as well as a reduction in GAGs accumulation, lysosomal mass, and mitochondrial-dependent oxidative stress, in a mutation-dependent manner. Noteworthy, MLPs allowed to obtain similar results to those observed with the conventional transfection agent lipofectamine. Overall, these results confirmed the potential of CRISPR/nCas9 as a genome editing tool for treating MPS IVA. We also demonstrated the potential use of MLPs as a novel delivery system for CRISPR/nCas9-based therapies.

Biomaterial-assisted targeted and controlled delivery of CRISPR/Cas9 for precise gene editing

“…A non-viral carrier based on CRISPR/nCas9-magnetoliposomes (MLPs) led to enhanced enzyme activity, and decreased accumulation of glycosaminoglycans (GAGs), lysosomal mass, and oxidative stress. 40 These findings demonstrated the potential for novel pH-responsive and cell-penetrating MLPs having a core–shell as a potential carrier for CRISPR/nCas9 delivery. The system was able to form complexes with CRISPR/Cas9 via the pH-responsive polymer.…”

Evidence of epigenetic landscape shifts in mucopolysaccharidosis IIIB and IVA

“…We confirmed the enzymatic activity defect in all evaluated fibroblasts, which agrees with previous reports using these cells. Interestingly, some cell lines also had reduced mRNA levels compared to healthy donor cells 29 , 34 , 35 , 52 . It is unlikely that the observed reductions in mRNA levels are explained by point mutations but rather suggest an additional mechanism regulating the levels of NAGLU and GALNS with a potential role in the pathophysiology of the diseases.…”

Evolution of CRISPR/Cas Systems for Precise Genome Editing

“…Cas9 nickases, mutated variants of Cas9, show higher efficiency in on-targets and lower effect in off-targets. Cas9n requires two sgRNAs to act [ 81 ]. Viruses produce anti-CRISPR proteins (Acr-p) to deactivate prokaryotic defense systems based on CRISPR systems.…”