Significant progress has been made over the first 24 months. We first focused on design and optimization of the BrainCRISPR nanoplatform for safety and functionality, and understanding how the different layers and components work together, to create progressively improved versions of the platform. We developed several prototypes for the inner core of the nanoplatform, formed from gold nanoparticles, insulin coating, CRISPR elements and other coatings for assist in intracellular trafficking, optimizing their design and ratio upon the particles for maximum effectiveness. We also designed the outer layer of the nanoplatform, with insulin alone or with other coatings to assist in crossing the blood-brain barrier, and antibodies to target specific brain cells. We confirmed that the coatings remained functional after being attached to the nanoparticles; different parameters, such as linker length and antibody binding strategies, were also found to be important for the nanoparticle’s functionality. Part of our work has been published in Journal of Nanotheranostics, and some is currently under review for publication. We next focused on the ability of the BrainCRISPR nanoplatform to overcome cellular barriers, its efficiency in gene editing and its safety profile in cells. We found that prototypes with insulin coating alone or together with a cationic polymer coating performed best in entry to cells and release of CRISPR components, and also showed high gene editing efficacy. We further demonstrated the prototypes' safety, showing they have low toxicity and low immunogenicity towards cell cultures. Taken together, these results are an important steppingstone towards a gold nanoplatform for safe and effective CRISPR gene editing for treatment of brain diseases.
