(M-487) Delivery of Cancer Specific CRISPR System using Fusogenic Lipid-coated Mesoporous Silica Nanoparticles

CRISPR-Cas9 and other engineered CRISPR systems have been powerful therapeutic strategies to cure many diseases by correcting disease-causing mutations in research. Nevertheless, clinical translation into human patient is not readily accessed due to lack of specific and efficient delivery system as well as potential concerns derived by viral delivery vectors that cause mutagenesis and unknown side effects. To overcome those limitations, non-viral delivery systems based on nanoparticles have attracted tremendous attention for efficient transport of CRISPR-Cas9 components to target cells. However, bypassing endosomal trapping is a critical step to avoid endosomal degradation that substantially reduces the gene-editing efficiency. Here, we present fusogenic lipid-coated mesoporous silica nanoparticles as a universal delivery platform of CRISPR-Cas9 proteins and sgRNA.

CRISPR-Cas9 protein and sgRNA was first mixed to form ribonucleoprotein (RNP), and then loaded into the pore cavity of mesoporous silica nanoparticles, which were then coated with a combination of lipids. Fusogenic lipids consist of cationic lipids, membrane fusion helper lipids, and pegylated lipids, which facilitates cytosolic uptake of RNP through membrane fusion bypassing endosomal degradation. In addition, we used ionizable lipids which is cationic at acidic tumor microenvironment while neutral at cytosolic region to facilitate efficient release of RNP payloads into cytosol. Delivery of RNP was assessed by gene editing at target locus by T7E1 assay and confocal imaging of fluorescence-tagged RNPs. To show anti-cancer therapeutic effect of RNP delivery, we delivered RNPs that induces DNA double strand breaks (DSB) at the specific mutational genomic loci of cancer cells, followed by cancer cell-specific death.

Confocal laser scanning microscopy imaging of fluorescent RNP delivery and gene editing at target locus by T7E1 assay analysis showed efficient cytosolic delivery of RNP and nuclear localization with our fusogenic nanoparticles. When RNP is directly loaded into fusogenic lipid, reduced amount of RNP was loaded for each lipid nanoparticle. Furthermore, RNP can be loaded both in and outside of liposome. Using mesoporous silica nanoparticles as a loading core overcomes such problems. Electrostatic interaction between negatively charged RNP and positively charged lipid potentially hinders sufficient release of RNP into cytosol. Application of Ionizable lipid can release RNP into cytosol at physiological pH after membrane fusion while maintaining membrane fusion ability outside of cell at acidic tumor microenvironment. Furthermore, modulating the ratio between cationic lipids and ionizable lipids could target tumor by selectively presenting positive charge for membrane fusion at acidic tumor microenvironment. Delivery of DNA damaging CRISPR-Cas9 RNP showed the cancer-specific death, which was assessed by cell viability assay and DNA damage visualized by nuclear puncta of immunostaining against γ-H2AX DNA damage marker. Application of mesoporous nanoparticles and fusogenic coating as a means of efficient cytosolic delivery platform for CRISPR RNP uptake will facilitate precision medicine by delivering specific CRISPR system and targeting specific genomic sequence depending on patient.