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Drug Delivery

Drug Delivery - Poster Session E

W15 - Optimizing a peptide-based nanoplatform for Cas9 ribonucleoprotein delivery and gene editing

Saturday, October 11, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology offers a promising approach for treating genetic diseases by enabling precise edits to DNA. Despite its potential, CRISPR-Cas9 faces significant delivery challenges due to its large size, net negative charge, and susceptibility to degradation. To address these challenges, various non-viral vectors have been explored to enhance delivery, such as lipids, polymers, and peptides. Among them, peptides have attracted interest due to their customizability, low immunogenicity, self-assembling capabilities, and long-term stability. Our lab has previously designed a fusogenic peptide, termed DIV3W, that responds to pH changes within the endosome, facilitating both endosomal escape and intracellular cargo delivery. The DIV3W peptide can also electrostatically interact with negatively charged nucleic acids, facilitating its binding to the RNP. This study aims to evaluate the DIV3W peptide for the delivery of Cas9 RNPs to induce efficient knockout of green fluorescent protein (GFP) in HEK293 human embryonic kidney cells.

Materials and

Methods: : To form the DIV3W:RNP complex, gRNA and Cas9 were combined in equimolar amounts to assemble the RNP. DIV3W peptide was then added at increasing molar ratios to form DIV3W:RNP complexes. To determine the minimum molar ratio for complex formation, an agarose gel electrophoresis assay was performed. Complexes were further characterized using dynamic light scattering (DLS) to assess particle size, dispersity, and charge. Cytocompatibility was then assessed using an MTS assay at varying molar ratios of DIV3W:RNP. After confirming cytocompatibility, cellular uptake was evaluated in HEK293 cells using fluorescence imaging and flow cytometry. A 96-hour gene knockout experiment was conducted to quantify the reduction in GFP signal in HEK293 cells mediated by the DIV3W:RNP complexes.

Results, Conclusions, and Discussions:: The DIV3W:RNP complexes successfully self-assembled at a minimum molar ratio of 20:1, as indicated by the white banding in the top of the gel (Figure 1A). This suggests that at the 20:1 ratio, the DIV3W peptide fully complexes with the RNP, which is further supported by DLS data demonstrating the formation of monodisperse, positively charged nanoparticles (Figure 1B). The 20:1 and 40:1 ratios of DIV3W:RNP exhibited a polydispersity index (PDI) below 0.3, indicating a uniform size distribution, which is important for maintaining stability. Additionally, the 20:1 and 40:1 complexes exhibited a zeta potential of 13.3 and 16.9 as well as a particle size of 124.6 nm and 141.9 nm, respectively, indicating the formation of stable, positively charged nanoparticles. Compared to the negative zeta potential of the free RNP, the positive charge of the DIV3W:RNP complexes suggests electrostatic interactions between the DIV3W peptide and the RNP. The positive surface charge may enhance cellular uptake by promoting interaction with the negatively charged cellular membrane, while the nanoparticle size is appropriate for cellular internalization.
To evaluate cytocompatibility with HEK293 cells, an MTS assay was conducted. The results confirmed that the DIV3W:RNP complexes were cytocompatible up to a 40:1 molar ratio, exhibiting cell viability above 80% (Figure 2). Based on these findings, uptake studies were conducted to further assess the ability of these complexes to enter cells efficiently. Initial results indicate around 60% uptake of the DIV3W:RNP complexes into HEK293 cells, confirming that the DIV3W peptide successfully facilitates RNP delivery. Therefore, to verify that the DIV3W:RNP complexes can induce gene edits, a GFP knockout study was conducted in HEK293 GFP cells. Preliminary studies suggest that the DIV3W peptide effectively disrupts the GFP gene to knockout GFP expression, particularly using 50 nM RNP complexed with DIV3W:RNP at a 40:1 molar ratio. These findings support DIV3W as a promising delivery platform for CRISPR-based therapies, advancing safe and effective genome editing approaches.

Acknowledgements and/or References (Optional): :