Kim Butler(1), Rita Serda(2), Achraf Noureddine(2), Ayse Muniz(3), Darryl Sasaki(1), Oscar Negrete(1), C Jeffrey Brinker(1,2)

1. Sandia National Laboratories
2. University of New Mexico Center for Microengineered Materials and the Department of Chemical and Biological Engineering
3. University of Michigan Biointerfaces Institute

CRISPR systems are versatile tools for genome editing and transcriptional gene regulation. While the majority of delivery vehicles for genome editing, including CRISPR, have been viral, there are limitations to these vectors including carcinogenicity and immunogenicity. Non-viral vectors have a lower immunogenicity profile than viral vectors, however, so far, non-viral vectors have demonstrated limited delivery efficiency, due to the size and complexity of the cargo (plasmid DNA or the Cas9 endonuclease/gRNA ribonucleoprotein complex (RNP)) and the need to deliver it to the nucleus of the target cell. To address the CRISPR delivery problem, we developed a CRISPR plasmid delivery system based on mesoporous silica nanoparticles (MSN) encapsulated within supported lipid bilayers (aka protocells). MSNs have tailorable pore size (2-20-nm) and intrinsically negative surface charge. Although the pore size is not large enough to accommodate plasmid DNA, we discovered that we can package negatively charged plasmids via fusion of cationic lipid vesicles on MSNs and that the MSN pore size is a major determinant of plasmid loading efficiency. Using dynamic light scattering, cryo-TEM, and high resolution confocal imaging, we have proven the plasmid-packaged protocells to be stable monosized colloids wherein DNA is co-localized with MSNs and protected within a supported lipid bilayer or multilayer. Protocells exhibit high levels of DNA loading and protection of DNA from nuclease degradation while maintaining a ~200nm hydrodynamic diameter and low polydispersity suitable for in vivo delivery. In vitro gene editing efficiencies were greater than 80%, equal to those seen with high dose lipofectamine, but with far lower toxicity. We hypothesize that the high gene editing efficiency is a consequence of spontaneous disassociation of the negatively charged DNA from the negatively charged MSN when the supported lipid bilayer is destabilized within the acidic endosomal environment following protocell internalization. To demonstrate CRISPR gene editing in a living system, we utilized the vascularized chick chorioallantoic membrane (CAM) model and intra vital imaging. Protocells successfully delivered CRISPR encoding plasmids to human A549 cells through the CAM vasculature resulting in expression of CRISPR Cas9 and editing of a reporter gene system after 24hours. Further, we demonstrated the same cationic vesicle packaging approach could also be used for RNP delivery.