Gene therapy represents a disease-modifying alternative to palliative treatments for neurodegenerative diseases (e.g., Parkinson’s, Alzheimer’s, and Huntington’s diseases). Idiopathic Parkinson’s has been linked to genetic mutations in GNDF, PRKN, and PINK-1 genes among others, associated with the Parkinsonian characteristic symptomatology. Over the years, several gene therapy strategies have been introduced, albeit still with considerable limitations. CRISPR/Cas9 offers several advantages for gene editing, including ease of implementation, versatility, and high-fidelity editing. Success of CRISPR/Cas9 therapy is strongly dependent on the deliver strategy of the various components without inducing significant immune responses while maintaining high bioavailability levels. An attractive route for improving the delivery efficacy is by transporting the gene cargoes aided by nanocarriers. Popular vehicles in this family include metallic nanoparticles, carbon-based nanomaterials, and lipid-based nanostructures. Our research group has dedicated significant effort to developing a nanoplatform for nucleotide delivery based on modified magnetite nanoparticles, which are attractive due to their strong magnetic responsiveness to guide the nanovehicles to the site of action. These delivery carriers contain a disulfide bond (pH-sensitive) and a potent translocating protein (Ompa) to facilitate internalization and promote endosomal escape. A plasmid containing sequences for both Cas9 nuclease and sgRNA (targeting the prior mentioned genes) was designed and conjugated to the nanoparticles aided by a short thiolated sequence. Neurons’ cytosol provides an environment where the disulfide bond is reduced to promote the CRISPR plasmid delivery. As a proof-of-concept experiment, we performed in vitro delivery of a conjugated Rhodamine-labeled cysteine, and confirmed delivery of 86% of the loaded material after two hours. Moreover, experiments in neuroblastoma cells revealed high endosome escape levels as evidenced by colocalization with Lysotracker below 11%. Future work will focus on testing genome editing in vitro in neuroblastoma, primary human astrocytes, and glial cell lines.

Delivery of CRISPR/Cas9 editing systems for the treatment of idiopathic Parkinson’s

ACS Fall 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.