(F-206) Peptide-Based Nanoplatform for Cas9/guideRNA Delivery and Gene Editing

Introduction:: Gene editing using CRISPR/Cas9 for the treatment of genetic diseases has rapidly advanced to become an integral part of the biotechnology field over the past decade. The CRISPR/Cas9 machinery consists of two key components: a guide RNA (gRNA) sequence that complements and pairs with the target gene, and a Cas9 nuclease that cleaves the DNA¹. The Cas9 and gRNA can be delivered via plasmids, mRNA, or ribonucleoprotein (RNP) complexes. RNPs overcome the need for transcription or translation and have demonstrated higher editing efficiencies and lower off-target effects than either plasmids or mRNA. However, due to their xenogeneic nature and charge properties, RNPs alone cannot be internalized into cells and therefore require a delivery vehicle. Viruses, lipids, and polymers have been used as vehicles for Cas9 RNPs, though limitations of safety and efficiency in delivering the gene-editing cargo into cells have posed a large hurdle to overcome¹. Peptides have emerged as a promising alternative for transporting bioactive nucleic acids into cells. We have previously demonstrated that a novel fusogenic peptide, DIV3W, successfully protects RNA cargo, is cytocompatible, and mediates efficient endosomal escape of bioactive RNAs². The objective of this study is to evaluate the DIV3W peptide for delivery of RNPs to induce efficient gene knockout.

Materials and Methods:: RNP:peptide complexes were synthesized by combining gRNA and Cas9 at an equimolar ratios, followed by complexation with various molar concentrations of DIV3W peptide. Complexation of the RNP with the DIV3W peptide was analyzed using agarose gel electrophoresis. To assess the cytocompatibility of the RNP:peptide complexes, RNP/DIV3W complexes were synthesized and incubated with HEK293 human embryonic kidney cells at increasing molar ratio complexes for 48 hours followed by an MTS assay to determine RNP:peptide cytotoxicity.


Results, Conclusions, and Discussions:: We observed that molar ratios greater than 1:1:20 (Cas9:gRNA:DIV3W) resulted in complete complexation of RNPs and therefore, demonstrates the ability of the peptide to self-assemble with RNPs (Figure 1A). Previous studies demonstrate that the DIV3W peptide is capable of protecting the RNP from premature degradation at molar ratios where complete complexation is observed. At a dose of 10 nM RNP, MTS results indicated that these RNP:peptide complexes were not cytotoxic to HEK293 cells, with viability greater than 90% for molar ratios up to 1:1:80 (Figure 1B). This result demonstrates the cytocompatibility of the DIV3W peptide and its potential for safe use in in vivo applications.
The DIV3W peptide can self-assemble with Cas9/gRNA to completely complex free RNPs. Additionally, the RNP/DIV3W complexes demonstrate cytocompatibility with HEK293 cells. These results demonstrate the potential of peptide-based delivery systems for Cas9 RNPs and warrant further investigation. Future work for this project includes characterization of the size and charge of the RNP:DIV3W complexes, evaluation of cellular internalization, and assessment of their gene editing capability.


Acknowledgements (Optional): : This work is supported by the National Science Foundation EPSCoR program NSF Award #OIA-1655740, National Science Foundation Faculty Early Career Development Program NSF Award #204669, and Clemson University’s Creative Inquiry program.

References (Optional): : 1) Zhang, S. et al. (2021), Theranostics, doi: 10.7150/thno.47007. PMID: 33391496; PMCID: PMC7738854


2) Samec, T. et al. (2022) Molecular Therapy - Nucleic Acids, doi: 10.1016/j.omtn.2022.09.012