Scientists from Zhejiang University have developed a novel delivery vehicle for CRISPR-Cas9 that is more effective than lipid nanoparticles (LNPs) in treating lung cancer. Rapid liquid nitrogen treatment can convert tumor cells into carriers for gene editing tools that target cancer in vivo. Cryo-shock eliminates tumor cell pathogenicity while preserving structure and surface receptor function.

In a mouse model of non-small cell lung cancer (NSCLC), this delivery system, loaded with CRISPR-Cas9, effectively targets the lung, ablating the tumor and extending survival time. Thanks to this liquid nitrogen cryo-inactivation technique, cells harvested from tumors that have been surgically removed, needle biopsied, or otherwise left behind can be frozen and used again as a vehicle for gene editing tools in the fight against cancer.

The research article, “Cryo-shocked tumor cells deliver CRISPR-Cas9 for lung cancer regression by synthetic lethality,” was published in Science Advances.

Cell-based gene editing delivery vehicles

The CRISPR-Cas9 genome editing system shows great promise as a tool for detecting and treating cancer, viral infections, and hereditary diseases. Problems with CRISPR-Cas9’s degradation or denaturation in the bloodstream and inefficient delivery are two obstacles to its wider clinical use.

One big problem with the current viral and nonviral CRISPR-Cas9 delivery vectors is that they can not accurately target specific tissues or cells. Furthermore, issues with immunogenicity, off-target gene effects, and dose-limiting toxicity prevent the further use of gene delivery vehicles such as viruses and LNPs in vivo despite their high gene editing efficiency.

Due to their homologous protein components, cell-based carriers show superior targeting capabilities compared to these synthetic and exogenous gene vectors. Unfortunately, the clinical use of certain types of living cells is limited due to the possibility of their physiological toxicity or pathogenicity.

Leveraging cryo-shocked cells as the targeting gene carrier

Feng Liu and Minhang Xin’s collaborative work shows that tumor cells can be inactivated and rendered harmless by quickly immersing them in liquid nitrogen while preserving their structural integrity. Maintaining the functional protein pool of cells treated with liquid nitrogen enhances their capacity to actively target the lungs through their interactions with endothelial cells. Additionally, cells treated with liquid nitrogen that have homologous receptors may improve drug delivery efficiency and homologous targeting by increasing the likelihood of interaction with tumor cells. Because liquid nitrogen treatment cells are bigger and have a higher electrical potential than cell-deprived vesicles, this CRISPR-Cas9 delivery system is more likely to be captured by pulmonary capillaries, which improves pulmonary retention.

Liu, Xin, and colleagues used a lung-targeted CRISPR-Cas9 drug delivery strategy to knock down cyclin-dependent kinase 4 (CDK4) in tumors, resulting in synthetic lethality in a KRAS-mutant mouse model of NSCLC. For in vivo CRISPR-Cas9 delivery, cryo-inactivated non-pathogenic KRAS-mutant NSCLC cells were used as a vector. This cell vehicle allows for highly targeted lung delivery via passive trapping by lung capillaries and cell interaction and adhesion mediated by CD44, thanks to the intact cellular architecture and preserved cell surface glycoprotein, CD44. While this cryo-inactivated cell delivery system could cause CDK4 ablation and the death of NSCLC cells with KRAS mutations, it did not affect normal cells.

Cells treated with liquid nitrogen retain tumor antigens, suggesting they may be useful as a vaccine in tumor immunotherapy. Accordingly, cells treated with liquid nitrogen may serve as vehicles for distributing immunomodulatory medications. Another advantage of liquid nitrogen treatment is that it allows for reusing cells obtained from abandoned tumors, postoperative resection, or needle biopsy.

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