Codelivery of CRISPR-Cas9 and chlorin e6 for spatially controlled tumor-specific gene editing with synergistic drug effects

Abstract

Controlled release of CRISPR-Cas9 ribonucleoprotein (RNP) and codelivery with other drugs remain a challenge. We demonstrate controlled release of CRISPR-Cas9 RNP and codelivery with antitumor photosensitizer chlorin e6 (Ce6) using near-infrared (NIR)– and reducing agent–responsive nanoparticles in a mouse tumor model. Nitrilotriacetic acid–decorated micelles can bind His-tagged Cas9 RNP. Lysosomal escape of nanoparticles was triggered by NIR-induced reactive oxygen species (ROS) generation by Ce6 in tumor cells. Cytoplasmic release of Cas9/single-guide RNA (sgRNA) was achieved by reduction of disulfide bond. Cas9/sgRNA targeted the antioxidant regulator Nrf2, enhancing tumor cell sensitivity to ROS. Without NIR irradiation, Cas9 was degraded in lysosomes and gene editing failed in normal tissues. The synergistic effects of Ce6 photodynamic therapy and Nrf2 gene editing were confirmed in vivo. Controlled release of CRISPR-Cas9 RNP and codelivery with Ce6 using stimuli-responsive nanoparticles represent a versatile strategy for gene editing with potentially synergistic drug effects.

In vivo delivery of CRISPR-Cas9 therapeutics: Progress and challenges

“…Deng et al. 131 co-delivered CRISPR-Cas9 RNP with photosensitizer chlorin e6 to a xenograft nasopharyngeal carcinoma mouse model using a near infrared (NIR) light and reducing environment-responsive polymeric nanoparticle. This delivery system was administered via repeated intravenous injection and encapsulated SpCas9/sgRNA RNP targeting nuclear factor erythroid 2-related factor 2 ( NRF2 ), an antioxidant regulator, for NHEJ-mediated disruption in CNE2 tumor cells.…”

Spatiotemporal control of CRISPR/Cas9 gene editing

“… 124 NTA-NPs loaded with Ce6 and Cas9/sgRNA are termed CC-NPs, which are then coated with the cationic polymer iRGD-PD to introduce tumor-targeted ligand iGPD and form new complex iGPD-CC-NPs (referred to here as T-CC-NPs). 124 With the help of tumor-targeted ligand iGPD, T-CC-NPs are accumulated on the tumor cell membranes and are internalized. Under NIR irradiation, Ce6 will generate reactive oxygen species (ROS), triggering the lysosomal escape of T-CC-NPs.…”

“…Under NIR irradiation, Ce6 will generate reactive oxygen species (ROS), triggering the lysosomal escape of T-CC-NPs. 124 Once the nanoparticles enter the cytoplasm, the high glutathione (reduced form) (GSH) level in the cytoplasm will cause rupture of the disulfide bond between NTA and PEG, allowing the release of Cas9/sgRNA, which then enters the nucleus for subsequent gene editing. 124 Another similar study designed a lactose-derived CRISPR/Cas9 delivery system by exploiting two internal bioresponsive stimuli: (1) asialoglycoprotein receptor (termed ASGPr), which is overexpressed on the surface of liver cancer cells and can specifically recognize the galactose residue on lactose, and (2) the breakage of disulfide linkages in a reducing environment.…”

Nanovesicle-Mediated Delivery Systems for CRISPR/Cas Genome Editing

“…Near-infrared (NIR) irradiation- and chlorin e6-triggered lysosome escape have been reported for the controlled release of Cas9 protein into the cytoplasm [ 134 ]. ROS generated by NIR irradiation in the presence of photosensitizers such as chlorin e6 are capable of destabilizing lysosome membranes, thereby releasing Cas9/sgRNA ribonucleoproteins from lysosomes.…”