Periodic Reporting for period 1 - BrainCRISPR (In Vivo CRISPR-Based Nanoplatform for Gene Editing: A New Disruptive Avenue for Non-Invasive Treatment of Genetic Brain Diseases)
Periodo di rendicontazione: 2022-06-01 al 2024-11-30
The revolutionary CRISPR/Cas genome editing technology has enormous potential for treating many genetic diseases. However, delivery of CRISPR machinery to diseased cells within the brain is one of the greatest challenges in medicine today. The ERC Consolidator-funded BrainCRISPR research project aims to develop a ‘smart’ gold nano-sized platform to safely and efficiently shuttle CRISPR machinery across the highly impenetrable blood-brain barrier, into deep brain regions and diseased brain cells, for effective genome editing. Our proof-of-concept results serve as the baseline of this pioneering research project, revealing the exceptional capabilities of insulin as a key to overcoming formidable brain and cell barriers. Within the project, the gold nanoplatform is designed with unique coatings of insulin and antibodies and is being thoroughly investigated and optimized to safely and efficiently deliver CRISPR to the brain without invasive procedures, and to be flexible and customizable, making it suitable for both general and patient-specific treatments. This comprehensive research will lead to a universal and adaptable nanoplatform and delineate design principles for its precise tailoring to specific needs of different brain diseases. This innovative BrainCRISPR nanoplatform can have a transformative effect on treatment of devastating genetic brain diseases.
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.
Based on the achievements accomplished during the project so far, we were able to make significant progress toward creation of the BrainCRISPR nanoplatform that could significantly enhance the fields of nanomedicine, gene editing, and brain disease therapy. Our preliminary findings suggest that we are on the right path to meet the long-term goals of the project and open new and promising directions of research that could lead to high impact publications and potential clinical translation. Using our systematic approach, we will continue to investigate and optimize our nanoplatform in brain disease models in culture and in mice. Altogether, we are confident that by the end of the project we will have an optimal BrainCRISPR nanoplatform, that enables non-invasive and targeted delivery, as well as high genome editing efficiency in the brain. Additionally, the program will provide fundamental knowledge and a set of modularity features and parameters to be adapted to a specific brain disease and specific patient requirements.