V13 - Peptide-based Nanoplatform for Cas9 Ribonucleoprotein Delivery

Introduction: : Clustered regularly interspaced short palindromic repeats (CRISPR) technology has transformed the field of genetic engineering over the last decade by enabling precise and permanent changes to the genome. The CRISPR system consists of CRISPR-associated (Cas) endonucleases that are directed to their target gene by a sequence-specific complementary guide RNA (gRNA) [1]. Despite its immense potential to correct and cure genetic diseases, CRISPR technology has been limited in its clinical translation. CRISPR/Cas9 can be delivered via plasmids, mRNA, or ribonucleoproteins (RNPs) [2]. Delivering Cas9 via RNP has demonstrated higher editing efficiencies and lower off-target effects than either plasmids or mRNA [3]. However, due to their large size, net negative charge, and endosomal entrapment, RNPs require a delivery vehicle for cellular internalization [3]. Peptides have emerged as a promising non-viral delivery platform for transporting bioactive nucleic acids into cells due to their high tunability, stability, and biocompatibility [4]. Our lab has previously synthesized and patented a novel fusogenic peptide, DIV3W, that can electrostatically complex with and protect RNA, is cytocompatible, and mediates efficient endosomal escape of bioactive RNAs [4]. This study aims to evaluate the DIV3W peptide for the delivery of RNPs to induce efficient green fluorescent protein (GFP) knockout.

Materials and

Methods: : RNP:DIV3W complexes were synthesized by combining Cas9 and gRNA at equimolar ratios, followed by complexation with various molar concentrations of DIV3W peptide. Successful complexation between the RNP and DIV3W peptide was analyzed using agarose gel electrophoresis. Size, polydispersity, and zeta potential characterization of both RNP and RNP:DIV3W nanoparticles was performed using dynamic light scattering (DLS). We investigated various concentrations of RNP (30, 40, and 50 nM) and molar ratios of DIV3W/RNP nanocomplexes (1:20, 1:40, and 1:60) to identify cytocompatible conditions for further studies. All complexes were incubated with HEK293 cells for 48 hours, followed by an MTS assay. Fluorescence imaging and flow cytometry measured cellular internalization of the nanoparticles with fluorescently tagged Cy5-siRNA in place of gRNA following a 4-hour incubation. To confirm DIV3W can facilitate endosomal escape, HEK293 cells were treated with DIV3W/RNP-Cy5 complexes, followed by immunofluorescent staining for early endosome antigen 1 (EEA1) at 30 minutes to demonstrate colocalization, and 4 hours to determine endosomal escape. Finally, RNP:DIV3W complexes were delivered to HEK293-GFP cells to knock out GFP expression. Changes in fluorescence after 96 hours were analyzed using fluorescence imaging and flow cytometry. Lipofectamine CRISPRMax was used as a positive control for uptake and knockout studies.

Results, Conclusions, and Discussions:: Complete complexation between the RNP and DIV3W was demonstrated at all molar ratios greater than 1:20 (RNP:DIV3W). DLS results show a size compaction and switch in zeta potential when RNP is complexed with DIV3W at the 1:40 molar ratio (Figure 1A). The shift from negative to positive confirms electrostatic complexation between the negatively charged RNP and positively charged DIV3W, leading to an improved interaction with the negatively charged cell membrane. MTS results confirmed that all RNPs were not cytotoxic to HEK293 cells up to the 1:40 molar ratio, with viabilities greater than 80% (Figure 1B). Flow cytometry results confirmed successful cellular internalization of Cas9/Cy5 siRNA RNP nanoparticles, with increased uptake at the 50 nM/1:40 molar ratio compared to Lipofectamine. Additionally, using the 50 nM/1:40 ratio for GFP knockout yielded a ~17% decrease in positive events and a 1.7-fold decrease in mean fluorescence intensity compared to the untreated control (Figures 2A, 2B). RNP delivery using DIV3W achieved editing efficiencies comparable to Lipofectamine, with no statistical significance between the two, demonstrating the effectiveness of the DIV3W peptide system.

The DIV3W peptide can self-assemble with and completely complex free RNPs to form monodisperse and positively charged nanoparticles. The RNP:DIV3W system is cytocompatible with healthy HEK293 cells up to the 1:40 molar ratio, highlighting its biocompatibility. Additionally, RNP:DIV3W nanocomplexes can successfully internalize into HEK293 cells and edit the target GFP gene at levels comparable to those of an industry-standard delivery system. These results demonstrate the early potential of peptide-based delivery systems for Cas9 RNPs and warrant further investigation. Future work of this project includes confirmation of endosomal escape and quantification of indel mutations in the GFP gene.

Acknowledgements and/or References (Optional):: [1] Jacinto FV, Link W, Ferreira BI. J Cell Mol Med. (2020)
[2] Li, T., Yang, Y., Qi, H. et al. Sig Transduct Target Ther 8, 36 (2023)
[3] Liu C, Zhang L, Liu H, Cheng K. J Control Release. (2017)
[4] Samec T., et al. Mol Ther Nucleic Acids. (2022)