The Life-Saving Delivery: How CRISPR and Nanotechnology can Treat Cancer
I love ordering things online, because I love getting packages in the mail (and I bet I’m not the only one!). But what if your order couldn’t be delivered? Maybe it didn’t fit in the box, or it couldn’t get to your house. And even if it did get to you, it might cause you harm.
This is one of the barriers to using CRISPR, a gene editing tool, to treat cancer. There just aren’t good delivery systems.
CRISPR can be used to enhance T cells and improve their cancer fighting abilities, or even to kill cancerous cells.
But in order to have CRISPR kill cancerous cells within our bodies, we need to make sure it is able to arrive at the tumor cells.
Delivering CRISPR is Hard
Because of CRISPR’s large size, current delivery methods have low editing efficiencies. Additionally, viral vectors potentially could cause carcinogenesis, immunogenicity, or insertions.
But nanoparticles could help solve that problem. Two types of nanoparticles have had promising results so far in delivering CRISPR to tumor cells: lipid nanoparticles and light-activated nanoparticles.
Lipid Nanoparticles
Lipid nanoparticles are appealing as a delivery method because they can both protect nucleic acids and escape from endosomes.
However, current commercially available lipid nanoparticles don’t have high transfection efficiencies. This could be because the plasmids used are too large, or because the nanoparticles don’t effectively package the plasmids, resulting in the overall compound being negatively charged and then repelled from the negatively charged cell membrane.
To utilize these nanoparticles for CRISPR-cas9 delivery to tumor cells, better methods of using them need to be developed. Fortunately, multiple new lipid nanoparticle delivery methods for CRISPR have been developed.
These methods can deliver either cas9 mRNA and the sgRNA, or plasmids containing the sequence for CRISPR-cas9 and the sgRNA.
Delivering cas9 mRNA and sgRNA
When using cas9 mRNA and sgRNA, the only components in the delivery complex are the payload and the lipids.
The cas9 mRNA and sgRNA, which are chemically modified to improve stability, are mixed with the positively-charged lipid nanoparticles to create a capsule.
Delivering Plasmids
In the case of encapsulating plasmids, additional steps are performed.
- First, the amount of space that the plasmids take up had to be minimized; this was done by first combining the plasmids with chondroitin sulfate.
- The chondroitin sulfate then helped the plasmids better bind to positively-charged protamine. This condenses the space the plasmid takes up while still keeping it stable.
- The core is then encapsulated by positively charged lipids.
- In this case, the lipids were also then coated with polyethylene glycol phospholipid, which helped the compound to be more stable and less toxic.
Both of these methods were found to be safe in mice. Treatment with lipid nanoparticles and cas9-mRNA/sgRNA in mice was able to achieve 84% and 91% editing efficiencies of PLK1 in glioblastoma and ovarian cancer cell lines, respectfully. (PLK1 codes for a kinase involved in mitosis and without it dividing cells can’t move past the G2 phase in mitosis and die.) Editing PLK1 resulted in the glioblastoma cells’ vitality being decreased 5 times, while the ovarian cells’ vitality was decreased by 10 times.
The plasmid-containing delivery complex was also able to inhibit tumor growth when targeting PLK1, although only a 3% PLK1 mutation rate was found. This inconsistency have been for a few reasons, such as if non-tumor cells were in the sample, or because of other factors that effect protein expression such as the processes of transcription and translation.
NIR Light Activated Nanoparticles
Photoreactive nanoparticles can be used to control when and where CRISPR is released. Using near-infrared (NIR) light in the tumor area, researchers are able to cleave bonds that then trigger the release of CRISPR-cas9. NIR light is appealing because it can penetrate the skin, but doesn’t damage tissue like ultraviolet light does.
Upconversion Nanoparticles
Upconversion nanoparticles (UNCPs) can take NIR light and convert it into ultraviolet light. This ultraviolet light can then be used to break bonds between photosensitive molecules and CRISPR, allowing for gene editing to take place.
The delivery system was created in four steps:
- The UNCPs are coated in a silica shell; this shell improves the biocompatibility and solubility.
- The compound is treated with a carboxylation reagent and then photocleavable molecules are attached.
- Cas9 proteins are bonded to the photocleavable molecules and then mixed with sgRNA to form complexes.
- Then the entire complex is covered with polyethylenimine to help it escape from endosomes.
Once inside the cell, these complexes can be activated by NIR light, and the molecule binding the CRISPR complex to the core is broken, allowing for CRISPR to be released to edit the cell’s genome.
In experiments, these complexes were found to not be harmful to cells when not editing essential genes. And when these complexes were used to target PLK1 in lung adenocarcinoma cells, the cells had a reduced viability. In mice, tumors treated with the UNCP complexes had tumors 74% smaller than in control mice after 20 days.
Codelivery with Ce6
Rather than directly freeing CRISPR, NIR light can also be used with a different delivery complex to trigger a chain reaction that ends with CRISPR being released.
For example, chlorin e6 (Ce6), a compound that creates reactive oxygen species (ROS), can be combined with CRISPR to kill cancer cells.
There are three steps to form the delivery complex.
- The copolymer NTA-SS-PEG-PCL and Ce6 first self assemble.
- A his-tagged CRISPR-cas9 and sgRNA complex is attached to the core.
- iRGD-PEG-pAsp(DAB), another copolymer, coats the complex.
When this complex enters the cell and is activated by NIR light, Ce6 is released and creates ROS. The cell releases the antioxidant glutathione, which then breaks the disulfide bond binding the Cas9 RNPs to the delivery molecule. The Cas9 RNPs are then able to disrupt the Nrf2 gene. Nrf2 is involved with the antioxidant response in cells, so by disrupting the gene the tumor cells weren’t able to protect themselves from the ROS.
When the researchers tested this in mice, 83% of tumor cells died and the mice lived 80% longer.
Challenges
There are still some challenges to using nanoparticles to deliver CRISPR.
Targeting
One of the challenges with lipid nanoparticles is getting them to the tumor cells. This can be done by injecting them at the site of the tumor, but this won’t work for many tumors. Therefore, we need a way for the treatment to be administered system-wide but still arrive at the right location.
In one study, researchers proposed using antibodies that would then bind to receptors on the cancerous cells to help get the nanoparticles to the tumor cells. In an experiment with mice, they were able to get an 82% editing rate of PLK1.
Additionally, in another study, researchers added the peptide iRGD to the delivery complex; this peptide targets tumor cells and can help deliver the complex.
Reaching Tumors with Light
To use nanotechnologies that utilize photo-sensitive nanoparticles to reach deeper tumors, the delivery complex will have to be adapted. Although NIR light can penetrate the skin, there is a limit to how deep it can reach. To reach deeper tumors, different molecules would be needed that could be activated with different wavelengths of light.
Affecting nearby cells
It’s also important that the lipid nanoparticles don’t accidently deliver CRISPR to non-tumor cells and edit their DNA. One proposed solution to this problem in cases where CRISPR mRNA is being delivered is to modify the mRNA so that in healthy cells RNA interference would prevent the mRNA from being translated.
Next Steps
To utilize lipid nanoparticles in cancer treatment, more research and trials must be done to further asses safety and efficacy. However, these new advancements are helping move towards a future of using CRISPR to treat cancer.
TL;DR
- CRISPR can be used to edit and kill tumor cells, but we need better delivery methods first.
- Nanotechnology can help us deliver CRISPR to tumor cells.
- Lipid nanoparticles can either deliver plasmids or mRNA, and have been both safe and effective in mice.
- NIR light can be used activate nanoparticles to then release CRISPR or other drugs that then lead to the release of CRISPR. These delivery systems have also been safe and effective in mice.
- Before these technologies can be widespread in cancer treatment, better targeting, light wavelengths for deeper tumors, and a way to not affect nearby cells need to be developed.
