How can Nanoparticles be used to deliver gene editing tools?
- Kevin
- Dec 25, 2022
- 4 min read
How can genes be edited?
Genes can be edited using a technology called CRISPR/Cas9. This technology uses microbial adaptive immunity to edit the genome in living cells. Originally, CRISPR/Cas9 is an adaptive immune system used by microorganisms to fight off viruses. Adaptive immune system refers to forming immunity to certain viral infections by utilizing information cells gained from previous exposure to pathogens. First, bacteria can store a fragment of the genetic materials from the virus in a farm of “spacer” in between CRISPR genes, which stands for Clustered Regularly Interspaced Short Palindromic Repeats. Bacteria then activates Cas9, an endonuclease that can cut DNA, and tracrRNA, RNA that is complementary to CRISPR genes, to create Cas9-crRNA complex, also known as an Effector Complex (crRNA refers to CRISPR RNA that are complementary to spacer sequences). Later, if the same virus invades bacteria and injects its genetic material, Cas9-crRNA complex will cut that genetic material if it complements the crRNA, ultimately killing the virus. Researchers determined to use this mechanism as a gene-editing tool. They first combined crRNA with tracrRNA to create single guide RNA (sgRNA), simplifying the process of introducing the CRISPR components to a target cell. This sgRNA can be easily modified to complement and target specific DNA sequences of interest. Then, once it reaches the target sequence, it will cut the sequence. This DNA damage induces either of the two repair mechanisms: Non-Homologous End Joining (NHEJ) or Homology-Directed Repair (HDR). NHEJ simply joins the cleaved ends of the cut DNA sequence. NHEJ is prone to eros, especially frameshift mutations occurring from insertion and deletions of genes, and it often takes place in the G1 phase of the cell cycle when there are not that many chromosomes to deal with. On the other hand, HDR involves the use of a “repair template”. Once the repair template is introduced to the target region, it will begin making edits to the sequence, ultimately repairing the damaged DNA sequence.
Image Credit: https://www.ncbi.nlm.nih.gov/books/NBK464635/figure/gen_edit.F2/
There are a few instances where CRISPR/Cas9 technology were implemented. For instance, this technology was used to create genetically modified animals. Researchers already successfully injected sgRNA and Cas9 mRNA directly into signe-cell embryos and generated genetically-modified mouse and monkey models, and this proves the vast potential of this technology for creating genetically modified animals where their genes can be altered for their better survival. Also, CRISPR/Cas9 technology has generated different disease models to study molecular mechanisms of various diseases and test new drugs and therapy, such as infectious disease models, cancer models, and neurological models.
How can gene-editing tools be delivered?
No matter how innovative or splendid CRISPR/Cas9 technology is, it is meaningless if we do not have a way to efficiently and precisely deliver the technology to the target region with no harm. Nanoparticles are perfectly suited for this role. Nanoparticles are very small engineered particles ranging from 1 nanometers to 100 nanometers in size. Because of their extremely small size (even smaller than the wavelength of visible light), they can pass through nearly all filters and do not reduce transparency of a material. Also, with a few additives on the surface of the nanoparticle, small particles, including Cas9 endonuclease and sgRNA, can be encapsulated in the nanoparticles so that they can be delivered in a relatively safe environment with extreme precision. Instances of nanoparticles being used to deliver gene-editing tools include the following:
Nanoparticles deliver CRISPR/Cas9 technology to the liver with higher efficiency to treat hyperlipidemia
Researchers at Tufts University and the Chinese Academy of Sciences used biodegradable lipid nanoparticles to encapsulate the gene editing tools (CRISPR/Cas9), which precisely target the cell and alter its DNA sequences. The lipid nanoparticles contain mRNA coding for Cas9 endonuclease and sgRNA that are necessary for gene editing. Since nanoparticles contain disulfide bonds, they will dissolve and release all the contents in themselves once they enter the cell body. Researchers applied this delivery method to mice to see if gene editing reduces the number of PCSK9 proteins associated with low LDL cholesterol in the liver. They could easily inhibit the production of PCSK9 in mice’s liver with 80% efficiency, proposing a possible treatment for hyperlipidemia.
Nanoparticles deliver CRISPR/Cas9 technology for fight against tumors
One of the most difficult challenges in tumor treatment is the fact that solid tumors are surrounded by thick, hardly penetrable cell walls and that these walls get thicker and thicker as tumors grow. However, lipid nanoparticles may be one of the solutions to this challenge. Dr. Siegwart from UT Southwestern Medical Center has been studying lipid nanoparticles to treat tumors in the human body. Dr. Siegwart and his research team had used already-existing lipid nanoparticles that are best suited for traveling to the liver, added gene editing tools, short interfering RNA (siRNA) in this case, that can inhibit production of focal adhesion kinase (FAK). Inhibiting the production of FAK will weaken the cell walls of the tumor and make it easier for the nanoparticles themselves to travel through the walls. It will also allow immune cells to enter the tumor and trigger immune responses. Moreover, they had used newly engineered nanoparticles to encapsulate CRISPR/Cas9 tools that can edit the gene PD-L1 producing PD-L1 proteins that inhibit the immune system from attacking tumors. They tested these nanoparticles in animal models with liver cancer. They first applied lipid nanoparticles with siRNA to block FAK production, then added newly engineered nanoparticles to alter PD-L1 gene. Not only the size of the tumor shrank to about one-eight the size of the original tumor, it also triggered more immune responses compared to the control (treated with empty nanoparticles).
Nanoparticles deliver Chemotherapy drug and a novel immunotherapy to treat Cancer
Researchers at University of Pittsburgh devised a novel immunotherapy which silences the gene that is responsible for immune-suppression of cancer cells. They then decided to use nanoparticles to test out the effectiveness of this therapy. They decorated the surface of the nanoparticles with chondroitin sulfate and PEG to help target tumors and avoid any health issues. Chondroitin sulfate and PEG can be helpful as they bind to receptors common to tumor blood vessels. This method showed a huge improvement: about 10% of nanoparticles reached the target in the mice model, while it was originally 0.7%. The new immunotherapy was tested in a mouse model of pancreatic cancer. The nanoparticles contain both FuOXP and siRNA, which will shut down Xkr8, which is responsible for distributing phosphatidylserine (PS) that acts as an immunosuppressant on the surface of the cell. The model treated with the nanoparticle showed better fighting environments against tumors, more immune responses, and decrease in tumor size.
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