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    Drug Delivery

    (M-481) Peptide Based Nanoplatform for Cas9/gRNA Ribonucleoprotein Delivery and Gene Editing

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row M - Poster # 481
    Introduction:: The CRISPR/Cas9 system is a rapidly evolving gene editing tool for treating genetic diseases1. The CRISPR/Cas9 machinery consists of a guide RNA (gRNA) sequence which complements the target sequence, and a Cas9 nuclease which cleaves the DNA1. The Cas9 and gRNA are typically delivered via plasmid, mRNA, or ribonucleoprotein (RNP) complexes. RNP complexes eliminate the need for transcription or translation once internalized within the cell, reduce off-target effects, and have a higher delivery efficiency compared to plasmid and mRNA delivery2. However, due to their large size, negative charge, and susceptibility to degradation within the endosome, RNP complexes require a delivery vehicle for successful internalization3. Peptides have emerged as a promising delivery system due to their low immunogenic response, high versatility, and self-assembling properties4. The fusogenic peptide, DIV3W, is able to electrostatically bind with the negatively charged RNP due to a large cationic tail. Our lab has previously demonstrated that DIV3W is biocompatible, able to facilitate endosomal escape, and protect RNA cargo4. The objective of this project is to evaluate the ability of the DIV3W peptides to deliver bioactive Cas9 RNP complexes and induce knockout of green fluorescent protein (GFP) in HEK293 cells. 

    Materials and Methods:: The RNP:DIV3W system is formed by spontaneous complexation of  Cas9 and gRNA at equimolar ratios, and then adding DIV3W at increasing molar ratios. The complexation of the RNPs and peptides was observed using an agarose gel electrophoresis assay. To analyze cytocompatibility, RNP:DIV3W complexes were formed and incubated with HEK293 human embryonic kidney cells for 48 hours, after which, an MTS assay was performed. The internalization of the complexes was visualized by forming RNP complexes using Cy5-labeled non-targeting siRNA (siNT) and Cas9 at equimolar ratios, and then adding DIV3W at varying molar ratios. The complexes were added to HEK293 cells and incubated for 24 hours, before analyzing using fluorescence imaging. To determine GFP knockout, GFP+ HEK293 cells were incubated with varying molar ratios of RNP:DIV3W complexes. The treated cells were incubated for 72 or 96 hours and knockout was visualized using a fluorescent microscope and quantified using flow cytometry.  

    Results, Conclusions, and Discussions:: We observed that the RNP/DIV3W complexes successfully self-assembled at a minimum molar ratio of 1:1:20 (gRNA:Cas9:DIV3W) as shown in the agarose gel shift (Figure 1A). The RNP/DIV3W complexes were cytocompatible with HEK293 cells at molar ratios up to 1:1:80 at a 10 nM concentration, demonstrating over 90% viability when analyzed via MTS (Figure 1B). To determine the optimal concentration of RNP for use with the DIV3W peptide, GFP+ HEK293 cells were treated with increasing concentrations of DIV3W complexed with RNP, from 10 nM to 50 nM, and left to incubate for 72 hours before visualizing GFP knockout. The 30 nM concentration demonstrated optimal GFP knockout and was used for subsequent studies. Fluorescent imaging revealed that the RNP/DIV3W complexes were efficiently internalized into HEK293 cells at a 30 nM concentration, with the highest internalization at molar ratios of 1:1:20 and 1:1:80 (Figure 2). 72 hour and 96 hour (Figure 3) treatments with the 30 nM RNP concentration demonstrated effective GFP knockout with varying DIV3W ratios. In conclusion, the results demonstrate that the DIV3W peptide is able to successfully self-assemble with RNP and the complex is cytocompatible with HEK293 cells. Furthermore, the DIV3W peptide facilitates internalization of RNP into HEK293 cells, resulting in GFP knockout. The initial studies have shown the promise of the DIV3W peptide delivery system for gene editing. Future studies for the project include quantification of cellular internalization and knockout, confirming cytocompatibility at higher treatment concentrations, identifying indel mutations to confirm knockout, and evaluating various routes of internalization. 

    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, Clemson University’s Spring CECAS UG Opportunity Grant #136. Clemson University’s Summer CECAS UG Opportunity Grant, and Clemson University’s Creative Inquiry program. 

    References (Optional): :
    1. Song, X. et al. Delivery of CRISPR/Cas systems for cancer gene therapy and immunotherapy.  Advanced Drug Delivery Reviews. 2021. ISSN 0169-409X, https://doi.org/10.1016/j.addr.2020.04.010. 
    2. Yip BH. Recent Advances in CRISPR/Cas9 Delivery Strategies. Biomolecules. 2020; 10(6):839. https://doi.org/10.3390/biom10060839  
    3. Wei, T., et al. Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing. Nature communications. 2020. 11(1), 3232. https://doi.org/10.1038/s41467-020-17029-3  
    4. Samec, T. et al. Fusogenic peptide delivery of bioactive siRNAs targeting CSNK2A1 for treatment of ovarian cancer. Molecular Therapy - Nucleic Acids. 2022. doi: 10.1016/j.omtn.2022.09.012