Controlling the KRas Oncogene With Crispr

Cancers cause millions of deaths every year. They are some of the deadliest diseases, and yet they are a product of our own human bodies, rather than some outside source.

What Causes Cancer?

Cancers stem from mutations in cells. Normally, cells are programmed to die as trying to keep a damaged cell would cost more effort than it would bring in. However, cells take damage over time, due to radiation and other phenomena. Usually, this would be fine, as different genes will tell the cell to kill itself if it is too damaged(tumor suppressor genes). However, sometimes a mutation happens that is not stopped, and thus it grows rapidly and uncontrollably. And that is how a cancer cell develops.

Usually, this is perfectly fine, as our bodies immune system would be able to get rid of these bad cells. However, as time progresses, more and more of these annoying little cells manage to escape detection and our immune system weakens more and more. This is why the risk of cancer goes further up the older you get.

Oncogenes are genes that will cause cancer as they allow for cells to divide and replicate uncontrollably. They stem from proto-oncogenes, which are just normal genes which aid in cell division and replication. Once a proto-oncogene is mutated, an oncogene can form.

Imagine a cell as a car and a proto-oncogene as the gas pedal. You can use it to accelerate, but you still have control. Now imagine that pedal gets stuck, and now you can’t stop accelerating. That is basically an oncogene!

Tumor supressor genes are essentially the opposite of oncogenes. Rather than accelerating cell division and growth, tumor supressor genes work to regulate and slow down cell division and replication. However, when these genes are tampered with, bad news is definitely about to come!

The most common abnormality to a tumor suppressor gene would be to the TP53 gene. Almost half of all cancers have mutations in this gene, and a lot of research has been done to see how we can activate these genes again!

Going back to the car analogy, the tumor suppressor gene acts as brakes, to stop the car when needed. Now imagine if the car had brakes that didn’t work! Now, that is a surefire way to get into a car wreck, and a cancer cell as well.

KRas

The Ras family of genes is responsible for 20 percent of all cancers. They are oncogenes, that were originally proto-oncogenes. However, the most dangerous form is KRas. Kras, being a proto-oncogene, normally acts to help cell growth. However, overexpression of this gene eventually leads to the development of cancer. KRAS-4B is the primary isoform(variant)in human cancers, and it causes almost 90 percent of all pancreatic cancers, around 30 to 40 percent of colon cancers, and around 20 to 15 percent of lung cancers.

Most ways of treating this overexpression of KRas includes using inhibitors to block its activation, thus stopping it. However, there are many other ways that could be explored to tackle KRas.

Using Crispr to Combat KRas

Due to its cheap cost and easy manufacturing, CRISPR(Clustered Regularly Inter-Spaced Palindromic Repeats) has become one of the most popular ways to edit genes. It’s alternatives(ZFNs and TALENs) do not offer the same cheapness and efficiency. Crispr is a powerful tool that is being leveraged to edit genomes, but it is still in its infancy. I thought of a great way to use this to combat KRas expression! By letting Crispr seek it out, and inducing a double-stranded break(DSB), Kras expression in cancer cells should cease to exist, and thus cancer growth will be halted significantly. But how can we get Crispr to a tumor?

Adenoviruses are being used in gene therapy to help deliver transgenes. By taking out harmful bits of DNA in their genomes, modified adenoviruses can carry the instructions to code for all sorts of genes. Adenovirus serotypes 2 and 5 are the most commonly used, and I hope to leverage a gutless Adenovirus five to carry the DNA to code for Crispr.

Did you just say a gutless virus?

Well, yes I did! Adenoviruses have gone through many generations of modification, but the most recent modification that we have made is to essentially wipe everything in their genome, except for the ITRs and a packaging signal.

Here the genome of adenovirus is shown. The normal genome is shown in black, and deletions are white. Insertions of transgenes are labeled in gray. https://www.nature.com/articles/3302612

By doing this, the gutless adenovirus also has been dubbed the helper-dependent adenovirus, as it needs another virus to replicate. To put it simply, another adenovirus will code for the whole entire virus(capsid, proteins, etc.) but it will not get it’s genome inserted inside as it has its packaging signal removed, thus allowing for it to not get packaged inside of a virus. If you want to learn more about these types of viruses, you can go here to check out my article on viral vectors as a whole.

Plasmid insert of DNA and gRNA that targets KRas. Generated via Benchling

Shown above, is how my gutless adenovirus will look like. It will contain the Cas9(Streptococcus Pyogenes)coding section, along with a section that will code for the KRas-targeting gRNA. This plasmid can be cloned either through polymerase chain reaction or through Gibson assembly.

Since this is only a plasmid sequence, I can only make guesses as to what this can do. First and foremost, the adenoviral vector carrying this sequence should be injected into an embolized tumor(tumor with no blood supply)that does have KRas oncogene activation. By cutting off the blood, we not only stop the tumor from growing, but we also minimize the chance that the vector travels elsewhere through the bloodstream.

Moreover, since this is a novel plasmid, it should be first tested in the lab in vitro(in glass/test tubes) before being used in clinical. trials.

Conclusion

As adenoviral vectors continued to be used and tested, and more Crispr variants begin to arise, there may be so many different things that we could never do. There are so many different viral vectors out there, and joining these two together could open the door to a world of other possibilities!

Thank you for reading this! I am Sam, and I am a TKS innovator that is really passionate about seeing how we can leverage gene editing to help in future space travel! You can check out my other medium articles about tech and mindsets by clicking on my icon down below. You can contact me through my LinkedIn or my email: samlitks2@gmail.com. Thanks again, and have a wonderful rest of your day!

I’m a TKS activator and I’m fascinated in understanding the mind and exponential technologies and how understanding both can lead to discovery and innovation!