Since the discovery of CRISPR and its application in biomedical research, it has undergone various iterative cycles of design optimization to address some of the limitations of off-target effects and editing efficiency. For large scale use in clinical therapeutics, research has focused on improving delivery methods to required cells. In a study published in Science Advances, a team of researchers from Tel Aviv University and Harvard Medical School have employed a lipid nanoparticle (LNP) delivery system to carry CRISPR components to cancer cells.
In vivo CRISPR to specifically target cancer cells has been proposed as an optimal therapeutic approach for aggressive and incurable cancers. From a library of ionizable amino lipids, the authors validated four CRISPR LNPs (cLNPs) for their ability to encapsulate and carry Cas9 mRNA and control GFP-sgRNA into the human cell line HEK 293. By assessing the percentage of cells with disrupted GFP expression and sequence, viability, and off-target effects in a variety of human cancer cell lines, the L8-cLNPs were deemed to be suitable candidates for specific gene editing in vitro with low toxicity.
To test the ability of LNPs to target cancer cells, specifically, the authors chose PLK-1 as the target gene. PLK1 is a cell cycle kinase essential for G2-M transition. Loss of PLK1 causes cell cycle arrest and cell death. sgPLK-1-cLNPs were able to edit up to 98% of HEK293 cells with <0.1% off-target editing. These cells were arrested in G2-M in 48 hours and were prone to cell death fivefold more than cells with control sgGFP-cLNPs, suggesting cLNPs have low toxicity in the absence of appropriate sgRNA.
The team analyzed the gene-editing of sgPLK1-cLNP in glioma cell lines and ovarian adenocarcinoma cell lines, which are difficult to treat. They observed PLK-1 disruption, cell cycle arrest, and reduced tumor cell viability in both lines. To confirm that the cLNPs themselves do not pose immune risks like toxicity and immunogenicity, the authors injected sgGFP-cLNPs into C57BL/6 mice and evaluated liver toxicity, blood count, and serum cytokine response 24h post-injection and found no significant differences with control mice.
The team also estimated the effective dosage of the sgPLK-cLNPs required to enable in vivo gene editing and inhibit tumor growth in glioma and ovarian cancer mouse models. A single intracerebral injection of sgGFP-cLNP into the mouse hippocampus could enable up to 72% gene editing in glioma, and only sgPLK1-cLNP could activate apoptotic pathways and increase median survival from 32 to 48 days in ovarian cancer. The sgPLK1-cLNPs specifically targeted tumor cells in the brain and not the terminally differentiated neurons in the glioma model.
EGFR Coating for Delivery Enhancement
To enhance cell-targeted delivery of sgPLK1-cLNP, the authors coated the cLNPs with anti-EGFR (epidermal growth factor receptor) since ovarian adenocarcinoma cells highly express EGFR and found that intraperitoneal injection of anti-EGFR coated c-LNPs could target the selected tumor tissue in mice and carry out gene editing. This is important to enable systemic administration of cLNPs to treat tumor cells specifically and limit side effects on healthy cells.
“This is the first study in the world to prove that the CRISPR genome editing system can be used to treat cancer effectively in a living animal,” said Dr. Dan Peer, leader of the study. “It must be emphasized that this is not chemotherapy. There are no side effects, and a cancer cell treated in this way will never become active again. The molecular scissors of Cas9 cut the cancer cell’s DNA, thereby neutralizing it and permanently preventing replication.”
The study used two cancer models that are very prevalent and difficult to treat-glioblastoma and metastatic ovarian cancer. The advantages of the cLNP delivery system are that it is non-viral, high editing efficiency (up to 98% in multiple cancer cells in vitro and up to 80% in vivo), low effective dosage, no toxicity, and improved tumor targeting, thereby increased safety. As a proof-of-concept, PLK1 is an ideal target for gene editing in certain cancers. However, the cLNP system can be developed for other tumor markers such as BCR and oncogenic mutations, such as RAS and p53.
By Sahana Shankar, Ph.D. Candidate
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