Prof. Lukas Dow, biochemistry, and his team published a study in Nature on Aug. 16, detailing a new, more specific gene editing tool that they created to study cancer mutations through preclinical mice models.
Dow began his research working with CRISPR-Cas9, another gene editing tool that targets specific DNA regions to mimic and study similar mutations seen in patient tumors.
“We actually know very little about how those specific mutations cause tumor growth and lead to therapy resistance or response,” Dow said. “So we have had a large, coordinated effort to try and understand those mutations that occur across many patients’ tumors.”
To accomplish his goal, Dow moved to optimizing base editing enzymes and developing a system that allows him to turn the expression of these enzymes on and off with doxycycline, an antibiotic also typically used to regulate gene expression. Dow used these enzymes to then create mutations in cells of animal models and compare them to cells without tumor mutations.
The gene editing tool utilizes technology from CRISPR-Cas9, according to Dow. The team combined Cas9, an enzyme that cuts DNA, and guide RNA, a type of RNA that determines which DNA region Cas9 cuts, with apolipoprotein B mRNA editing enzyme, catalytic polypeptide — an enzyme commonly known as APOBEC that creates single base mutations in DNA.
Dow explained that the guide RNA takes Cas9 to a specific region in the genome, allowing Cas9 to then cut one of the DNA strands at the targeted site. Afterwards, APOBEC changes the nucleotide bases in the DNA, typically changing cytosines to uracils or thymines. Additionally, the gene editing tool has a domain called the uracil glycosylase inhibitor, which prevents the newly changed bases from being reverted back to cytosine.
However, Dow and his team encountered some challenges during the process. For example, frequent expression of APOBEC resulted in unwanted and random mutations of various RNA molecules in cells. These essential RNAs could potentially become translated into dysfunctional proteins that are detrimental for the cell.
To address this, a single gene copy that produces APOBEC is integrated into the cell’s genome and controlled by gene expression regulator doxycycline in order to determine how much APOBEC is produced.
In addition, the gene expression of APOBEC varies among different types of cells. Not all cells in the intestine, liver and pancreas produced this enzyme when incorporated into their genome. Thus, Dow inserted two copies of the gene in these cells instead of one to compensate for the lower expression of APOBEC.
Dow also encountered difficulties delivering components of the gene editing tool, or doxycycline, into certain tissues such as the brain. Dow proposed that various other delivery methods could be used in lieu of the gene editing tool, such as using viruses or zapping cells to open up the cell membrane. On the contrary, doxycycline could be directly injected into the brain in order to activate the gene editing tool.
Despite these difficulties, the gene editing tool presents favorable opportunities to understand the effects of single-base genetic changes on tumors through models and determine which therapy is effective against cancer. It could also be used as treatment itself by editing and reverting genetic changes observed in tumors. The tool could even be used to study other disorders outside of cancer.
“There are ways that the gene editing tool can be used as both therapy, as well as development of models to understand disease,” Dow said. “If you’re doing it in a clinical setting, there are a range of different editing enzymes that allow you to create different types of mutations.”
Dow plans to continue his projects involving the different applications of base editing in cancer biology. He is currently creating a library of guide RNAs that help induce hundreds and thousands of mutations found in tumors. Additionally, he is creating models in lung, colon and pancreatic cancers in order to further study the effects of tumor mutations.