The Silencing of CDKN3 to Treat Kidney Cancer
When was the last time you went to the bathroom? I’m guessing probably not very long ago. It must’ve felt good to get all of the waste collected from your bloodstream out of your body. And you have your kidneys to thank for that — they’re the organs that filter waste from your body to be released.
It would be the worst if something were to happen to them…something like cancer.
As you’re reading this article, someone is about to die of cancer. Cancer is a disease where your cells multiply at an exponential rate. One of the world’s most deadly diseases, cancer can spread throughout your whole body and destroy the function of your organs and your kidneys are no exception.
Many cancer therapies exist but a lot of them destroy healthy cells while also destroying tumor cells. That’s where the use of oncolytic virotherapy proves to be the most precise cancer therapy, if of course, engineered correctly. To learn more about oncolytic virotherapy, you can read the article I’ve written on the subject here.
But in case you don’t feel like reading the full article, the basic idea behind oncolytic virotherapy is that non-pathogenic strains of viruses like the flu and herpes can be engineered to deliver gene therapies precisely and effectively to cancer cells!
This article will detail the idea I have for a oncolytic virotherapy specific to kidney or renal cancer. Read more to find out!
The Basics of Kidney Cancer:
Kidney cancer is a type of cancer that, well…starts in the kidney! As already mentioned, renal cell carcinoma is the most common type of kidney cancer in adults.Specfically it targetst the renal tubes, which are the part of the kidney that clean your blood of waste to make urine.
Clear cell renal cell carcinoma is the most lethal renal tumor commonly found in adults, which gets its name due to its clear appearance as viewed through a microscope.
Research was done by Wei Wenhao and his team last March to identify some of the genes involved in cell cycle regulation, and how overexpression or underexpression of these genes affects the rate at which renal cell carcinomas develop.
CDKN3 is the target gene for this therapy, and it is one of many genes that regulate the cell cycle. The research found that the overexpression of CDKN3 in clear cell renal carcinoma is linked to metastasis, which is the growth and spreading of cancer, of renal cancer.
My Experimental Idea:
The main idea of this therapy: Gene silencing of CDKN3 through the viral injection of an RNA interference (RNAi) construct, a siRNA construct which is complementary of the target gene, to be engineered against CDKN3.
This would be done to further observe the role of CDKN3 in carcinomic cell regulation. The hope is, if done correctly, this could be offered as another kidney cancer therapy.
The Main Parts:
- The viral carrier: Newcastle Disease Virus
- The gene carrier and tumor specific ligand
- siRNA construct and RNA interference
Now we’re going to take a look at the system part-by-part!
The viral carrier: Newcastle Disease Virus
Gene therapies need to have some way to get to the target cells, and one way to do this is through a viral carrier. Viral vectors are engineered through the process of molecular cloning to not inject any of their own DNA into the host cell, so as to improve the efficacy of the delivery system, and of the therapy as a whole.
Newcastle Disease Virus (NDV) is a Paramyxovirus that causes a deadly disease in birds. In humans, it causes conjunctivitis or some flu-like systems. Through the power of genetic engineering, we can take non-pathogenic strains of NDV and modify them to help treat cancer. It’s already been shown to be a promising oncolytic virotherapy.
A non-pathogenic strain of the Newcastle virus would be modified with an F protein from the virus’ own genome that fuses easily with NDV. The F protein would be modified with a multibase cleavage and activation site to enhance its anti-tumor properties. When the F protein is added to the Newcastle virus, it proves to be an effective vector.
The tumor-specific ligand:
The tumor-specific ligand that would be attached to the Newcastle disease is a combination of a gene carrier known as PEA and a cytotoxic agent. PEA is a gene carrier that has a high transfection efficiency. The cytotoxic agent is a peptide with the sequence LTCQVGRVH. It specifically targets kidney cells and is bonded to PEA through an amide bond between the amine group of PEA and the carboxyl group of the peptide.
Tumor specificity is a huge issue in oncolytic virotherapy because the viral vector has to go through compounds in the bloodstream that often obstruct its path to the target tumor; having the tumor specific ligand accounts for that as well as increases accuracy.
RNA interference:
Imagine that you were able to switch your genes on and off — just like a light switch, right? RNA interference is that process of switching a light on and off, and the RNA interference construct, or RNAi, is that switch.
RNA interference is the process of using single-stranded RNA (siRNA) to bind to a gene to stop its function completely — literally turning it off. If this could be applied in a long-term manner, we can halt cancer in its path and potentially stop it from continuing to grow and spread.
The diagram above shows the process of RNA interference, and you’ll notice that it functions a lot like CRISPR-Cas13, which cuts up RNA!
After the RNase cuts the double-stranded RNA (dsRNA), it creates siRNA that bind with the AGO enzyme to form the RISC complex. The RISC complex uses the siRNA as guide RNA (gRNA) to locate the target sequence of mRNA. RISC then cuts the mRNA, which leads to the gene silencing.
But that’s what occurs in nature — how do we synthetically induce RNA interference?
The actual RNA interference works the same, but the siRNA has to be generated on it’s own. We take our target sequence and we engineer a 21–23 base pair sequence complementary to the target DNA sequence. The idea is that when the CDKN3 is transcribed from DNA into mRNA, the synthetic siRNA combined with the naturally occuring AGO enzyme would form the RISC complex and induce the RNA interference for CDKN3.
We can talk a lot about the parts, but how would the experiment actually work ? Let’s take a look.
To sum up the process…
- We engineer the PEA to containing the siRNA through molecular cloning (which you can read about in my article here)
- We attach both the PEA with the siRNA into the XbaI site of the NDV, which provides a site for the receiving of outside genetic material into the virus.
- We’d then attach LTCQVGRVH to the capsid of the nonpathogenic strain of Newcastle virus to serve as a flashlight to find the exact renal carcinoma.
- The virus transfects its material, including the siRNA, into the in vitro tumor cells, and we observe RNA interference!
Future steps
The next steps would be to take this to a lab setting and test these ideas to better observe the effects of this therapy and to use gene silencing to observe the role of CDKN3.
Although CDKN3 was chosen for this experiment, it’s important to note that there are a multitude of other genes that affect the metastasis of clear cell renal cell carcinoma. Although a similar study has been done to observe the roles of those genes in the metastasis of clear cell renal cell carcinoma, similar gene silencing experiments would be necessary to observe how all of these genes work in tandem.
If gene silencing of all of the genes is proven to be effective in reducing the growth of clear cell renal cell carcinoma, then it would be possible to develop a marketable product to help slow the metastasis and potentially even stop clear cell renal cell carcinoma in its tracks. Kidney cancer sufferers everywhere would live kidney cancer-free lives — and go to the bathroom in one piece!
Thank you for reading my article on this cancer gene therapy! If you are interested in helping me develop this further, or know someone who would be interested, you can contact me on Linkedin, or through my email: writetoapurva@gmail.com. Until next time!
