Development of a non-viral delivery nanoplatform for genomic therapy based on iron oxide nanoparticles and recombinant Cas9 for potential use in genetic heterozygous orphan diseases

Abstract

Since it was first described, the CRISPR/Cas9 technology has opened new possibilities in the molecular biology fields. CRISPR/Cas9 has become one of the favorite tools for researchers from cell culture improvement to design of therapies for rare diseases. However, its clinical application has been hindered by the dearth of safe and efficient delivery systems. Here, we develop and characterized a magnetite-based nanoplatform to potentially deliver, both recombinant Cas9 and single guide RNA (sgRNA) targeting a pathogenic COL3A1 mutation in a cell culture model of hereditary Thoracic Aortic Aneurism. Specifically, we designed and synthesized several sgRNAs to test their activity through an allele specific DNA digestion assay. For this, we amplified the affected region using genomic DNA from a patient carrying the mutation and evaluated the cleavage efficiency of different Cas9-sgRNA ribonucleoproteins. Once the optimal conditions were obtained, we carried out DiGenome-Seq analysis to identify potential off-target effects. In parallel, we synthesized Magnetite Nanoparticles (MNPs), functionalized the particles by adding a polymer spacer (polyethylene glycol PEG) and conjugated the cell-penetrating peptide (CPP) Buforin II (BUFII) for membrane translocation alongside recombinant Cas9. We successfully characterized the MNP-PEG-BUFII-Cas9 conjugates by transmission electron microscopy (TEM) and dynamic light scattering (DLS), finding an average particle size of 15 nm and a hydrodynamic diameter from 120 to 250 nm. Fourier transform infrared spectroscopy (FTIR) lead us to corroborate correct functionalization in each step and find N-H stretch peaks at 3180 cm ¹ and 3500 cm ¹ which corresponded to the Cas9. And thermogravimetric analysis (TGA) displayed rising weight losses (6.97%, 7.77%, 10.41%) in each step of the synthesis. We also conducted a biocompatibility battery test that includes lactate dehydrogenase (LDH) cytotoxicity assay, platelet aggregation, hemolytic activity, and the Ames test for mutagenicity (Salmonella Tiphimurium TA98 strain). We demonstrated in vitro biocompatibility and highlight a platelet aggregation behavior (p > 0.001) of MNP-PEG-BUFII-Cas9. Finally, we performed a proof-of-principle of our MNP-PEG-BUFII-Cas9 nanoplatform, evaluating cell (HDFa and HaCat) internalization and endosomal escape of the MNP-PEG-BUFII-Cas9 without the sgRNA by confocal microscope image analysis. Overall, here we demonstrate the efficacy of MNP-PEG-BUFII-Cas9 nanoplatform as safe and promising non-viral delivery vehicles for CRISPR/Cas9 localized gene editing attempting to treat heterozygous mutations.