(W1230-03-17) Peptide-Based Nanoplatform for Improved Cas9 RNP Delivery and Editing

Purpose: 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 complimentary guide RNA (gRNA).¹ Despite its immense potential to correct and cure human genetic disease, CRISPR technology has been limited in its clinical translation. The Cas9/gRNA system can be delivered to cells in multiple ways, including plasmids, messenger RNA (mRNA), or ribonucleoproteins (RNPs). Of these platforms, RNPs have increased gene editing efficiencies and transient expression, reducing the risk of off-target effects.^2,3 However, due to their large size, net negative charge, and poor endosomal escape, RNPs require a delivery vehicle for efficient cellular internalization to achieve therapeutic efficacy.⁴ Peptides have emerged as attractive vehicles for CRISPR-Cas9 delivery, as they are highly tunable, exhibit high biocompatibility, and have demonstrated great success in enabling endosomal escape of bioactive cargo.^5,6 Our lab has previously designed a novel fusogenic peptide, termed DIV3W, that can electrostatically complex with and protect nucleic acids, is cytocompatible, and mediates efficient endosomal escape of bioactive nucleic acids.⁷ Therefore, the objective of this study is to evaluate the DIV3W peptide for delivery of RNPs to induce efficient reen fluorescent protein (GFP) knockout in healthy HEK293 cells.

Methods: DIV3W:RNP complexes were synthesized by combining gRNA and Cas9 at equimolar ratios, followed by complexation with various molar concentrations of DIV3W peptide. Complexation between RNP and DIV3W was analyzed using agarose gel electrophoresis and was further characterized using dynamic light scattering (DLS). MTS assays determined the cytocompatibility of DIV3W:RNP complexes at varying molar ratios. To visualize cellular internalization, gRNA was replaced with Cy5-fluorescently-tagged siRNA to visualize and quantify 4-hour internalization using fluorescent imaging and flow cytometry. DIV3W:RNP and Lipofectamine:RNP complexes were delivered to HEK293-GFP cells to knockout GFP expression, and changes in fluorescence after 96 hours were analyzed using flow cytometry.

Results: DIV3W:RNP molar ratios greater than 20:1 resulted in complete complexation as shown by a lack of free gRNA or RNP banding and is therefore, a successful delivery vehicle capable of protecting the RNP from premature degradation (Figure 1A). DIV3W:RNP molar ratio complexes were measured to be ~140 nm with a PDI < 0.3, confirming monodisperse nanoparticle formation (Figure 1B).  Additionally, DIV3W complexation was further supported through a switch from a negative zeta potential of the RNP to a positive zeta potential at the 20:1 and 40:1 molar ratios. MTS results demonstrated that all molar ratios were above 80% viability, except for the 60:1 DIV3W:RNP at a 50nM RNP concentration, confirming cytocompatibility with HEK293 cells (Figure 2). Preliminary uptake studies show ~50-70% cellular internalization after 4 hours. Knockout studies have demonstrated significant GFP knockout using specifically the 40:1 DIV3W:RNP molar ratio with 50 nM RNP concentration as compared to Lipofectamine CRISPRMax, highlighting comparable editing efficiencies to a commercially available system (Figure 3).

Conclusion: The DIV3W peptide can self-assemble with and completely complex free RNPs and is cytocompatible with HEK293 cells. The DIV3W:RNP nanocomplexes can escape the endosome prior to lysosomal degradation and traverse the nuclear membrane, as shown through successful gene editions. This peptide system has achieved significant GFP knockout comparable to that of Lipofectamine. These results demonstrate the potential of peptide-based delivery systems for Cas9 RNPs and warrant further investigation.

References: (1) Hossain, M. A. CRISPR-Cas9: A Fascinating Journey from Bacterial Immune System to Human Gene Editing. In Progress in Molecular Biology and Translational Science; 2021; Vol. 178. https://doi.org/10.1016/bs.pmbts.2021.01.001.
(2) Yip, B. Recent Advances in CRISPR/Cas9 Delivery Strategies. Biomolecules 2020, 10 (6), 839. https://doi.org/10.3390/biom10060839.
(3) Sioson, V. A.; Kim, M.; Joo, J. Challenges in Delivery Systems for CRISPR-Based Genome Editing and Opportunities of Nanomedicine. Biomed Eng Lett 2021, 11 (3), 217–233. https://doi.org/10.1007/s13534-021-00199-4.
(4) Wang, S. W.; Gao, C.; Zheng, Y. M.; Yi, L.; Lu, J. C.; Huang, X. Y.; Cai, J. Bin; Zhang, P. F.; Cui, Y. H.; Ke, A. W. Current Applications and Future Perspective of CRISPR/Cas9 Gene Editing in Cancer. Molecular Cancer. 2022. https://doi.org/10.1186/s12943-022-01518-8.
(5) Samec, T.; Boulos, J.; Gilmore, S.; Hazelton, A.; Alexander-Bryant, A. Peptide-Based Delivery of Therapeutics in Cancer Treatment. Mater Today Bio 2022, 14, 100248. https://doi.org/10.1016/j.mtbio.2022.100248.
(6) Alexander-Bryant, A. A.; Zhang, H.; Attaway, C. C.; Pugh, W.; Eggart, L.; Sansevere, R. M.; Andino, L. M.; Dinh, L.; Cantini, L. P.; Jakymiw, A. Dual Peptide-Mediated Targeted Delivery of Bioactive SiRNAs to Oral Cancer Cells in Vivo. Oral Oncol 2017, 72, 123–131. https://doi.org/10.1016/j.oraloncology.2017.07.004.
(7) Samec, T.; Alatise, K. L.; Boulos, J.; Gilmore, S.; Hazelton, A.; Coffin, C.; Alexander-Bryant, A. Fusogenic Peptide Delivery of Bioactive SiRNAs Targeting CSNK2A1 for Treatment of Ovarian Cancer. Mol Ther Nucleic Acids 2022, 30, 95–111. https://doi.org/10.1016/j.omtn.2022.09.012.