Development of Non-Viral Vectors for Neuronal-Targeting of Crispr as a Therapeutic Strategy for Neurological Disorders

Biomedical research has classified various targets for gene therapy that can halt or reverse the progression of neurodegenerative pathologies, however, the translation of such therapeutics to the clinic has been limited. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system have been the focus of intense research in the last decade due to its superior ability to desirably target and manipulate DNA sequences in vitro and in vivo. A main challenge is the lack of a clinically translatable system to specifically deliver CRISPR-Cas to target cells while minimizing its presence in other tissues. Non-viral delivery systems represent an attractive approach to developing gene-based therapeutics due to their ability for specific targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis. Here, we describe the development a peptide-based delivery system that can efficiently deliver CRISPR to neuronal cells for therapeutic genome editing. By comparing different cell-penetrating peptides for their ability to deliver a CRISPRencoding plasmid or RNA-protein (RNP) to cells, we found that the amphipathic PepFect14 was of comparable efficiency to commercial transfection reagents. On the other hand, cationic poly-arginine peptides did not achieve sufficient functional uptake to edit a genomic target. Efficiency was evaluated in reporter cells and wild-type cells by T7E1 assay. We developed the neuronal-targeting C2-PF14 peptide by fusing a neuronal-targeting sequence, C2, to PepFect14. C2-PF14 showed significant preferential uptake of CRISPR-Cas9 components to neuronal SH-SY5Y cells, compared with non-neuronal S2103 cells. Furthermore, RNP/C2-PF14 complexes formed nanosized, monodispersed, non-toxic nanoparticles capable of performing gene editing at a target genomic location. Reporter assay and immunocytochemistry analyses showed that C2-PF14 achieved higher efficiency of RNP delivery to SH-SY5Y cells than Lipofectamine RNAiMAX. Finally, we show that RNPs delivered to SH-SY5Y cells by C2-PF14 edited SNCA gene by T7E1 assay and western blot, and relieved Parkinson’s disease (PD)-associated MPP+-induced toxicity in neuronal cells in vitro. This represents a proof-of-concept towards the development of a safe in vivo braintargeted genome-editing strategy for the treatment of PD and other neurological disorders.