For the first time, researchers safely and effectively treated cells and mice with ovarian cancer using the CRISPR-Cas9 gene-editing tool plus a newly developed lipid nanoparticle (LNP) delivery system, which targeted and destroyed the cancer cells by genetic manipulation.
This new therapeutic strategy has the potential to treat not only other types of cancer, but also genetic diseases and chronic viral infections, the researchers said.
The findings were described in the study, “CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy,” published in the journal Science Advances.
CRISPR-Cas9 is a molecular tool that can be used to precisely edit DNA in cells and has the potential to become a cancer treatment by permanently disrupting genes associated with tumor survival.
This strategy would overcome some of the limitations of traditional therapies and improve treatment efficacy with fewer treatments. However, the current systems that deliver CRISPR-Cas9 result in low gene-editing efficiencies.
Lipid nanoparticles (LNPs) are tiny fatty spheres that are clinically approved to deliver molecular therapies to diseased cells.
Current LNPs are optimized for small molecules, but not for those needed to deliver CRISPR-Cas9 components, which are larger. Furthermore, while several delivery vehicles for CRISPR that target the liver have been reported, formulations designed for other tissues were less efficient.
“The CRISPR genome editing technology, capable of identifying and altering any genetic segment, has revolutionized our ability to disrupt, repair, or even replace genes in a personalized manner,” Dan Peer, PhD, the study’s senior author, said in a press release.
“Despite its extensive use in research, clinical implementation is still in its infancy because an effective delivery system is needed to safely and accurately deliver the CRISPR to its target cells,” added Peer, also a professor at Tel Aviv University (TAU), in Israel.
In collaboration with investigators in the U.S., researchers at TAU overcame these challenges by redesigning LNPs to deliver the larger CRISPR-Cas9 molecules. These newly designed LNPs were tested in cells and mouse models of metastatic ovarian cancer as well as glioblastoma, the most aggressive type of brain cancer.
First, the team found a specific fatty acid that was able to encapsulate the two molecules needed for CRISPR-Cas9. One, the Cas9 messenger RNA (mRNA), is a molecule that carries the information for the Cas9 enzyme, while the other, single-guide RNA (sgRNA), matches and binds to a specific DNA sequence that is spliced by Cas9.
As a proof-of-concept, the chosen sgRNA targeted a gene called PLK1, which codes for a protein that is critical for healthy cell function. Disruption of this gene stops the cell cycle and kills growing cells.
Incubating a standard cell line with an LNP containing this sgRNA — called sgPLK1-cLNP — resulted in a fivefold decrease in cell viability compared with untreated cells or those given an LNP targeting a different non-essential gene.
Cell viability was preserved after control LNP treatment, which suggested that CRISPR-LNPs “may have low toxicity at therapeutically relevant doses,” the researchers wrote.
This experiment was replicated in a human metastatic ovarian cancer cell line (OV8) that is highly resistant to chemotherapy. Compared with control cells, there was a tenfold drop in OV8 viability. The percentage of living glioblastoma cells also was reduced fivefold after treatment.
To test this system in an animal model of ovarian cancer, the sgPLK1-cLNP was coated with antibodies that bind the EGFR protein, which is highly expressed on the surface of OV8 tumors. These antibodies helped guide the LNP to the proper cell type.
Mice bearing OV8 tumors were injected with coated sgPLK1-cLNPs, and after two days, tumors were collected and the cells were analyzed. A total of 82% of the tumor cells were edited at the PLK1 gene. Meanwhile, less than 1% of PLK1 gene editing was detected in cells from control mice.
Further treatment of OV8 mice with sgPLK1-cLNP strongly inhibited tumor growth and led to an 80% increase in overall survival. About 90% of treated animals were alive after 60 days, whereas none of the controls survived that long. In parallel, sgPLK1-cLNP in a glioblastoma mouse model also was effective at editing the PLK1 gene. It increased overall survival by 30%, the results showed.
In normal mice, additional tests found this LNP system did not show clinical signs of toxicity or potentially dangerous immune responses.
“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,” Peer said.
“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,” he said.
“The molecular scissors of Cas9 cut the cancer cell’s DNA, thereby neutralizing it and permanently preventing replication,” Peer added.
While these results demonstrated the technology’s potential in ovarian cancer and glioblastoma, the researchers noted that it could be used to treat other types of cancer, rare genetic diseases, and even chronic viral diseases such as acquired immunodeficiency syndrome (AIDS).
“We now intend to go on to experiments with blood cancers that are very interesting genetically, as well as genetic diseases such as Duchenne muscular dystrophy,” Peer said. “It will probably take some time before the new treatment can be used in humans, but we are optimistic.”
“I believe that in the near future, we will see many personalized treatments based on genetic messengers – for both cancer and genetic diseases,” Peer added.