Help Not Harm: How Viruses Can Treat Cancer

As a kid, I remember hating getting a flu shot.

There was something about the sharp needle that terrified me to absolutely no end. And I had to get it every. Single. Year. That blew (and still blows) my mind! Every year, there are over three million cases of the flu, and 57% of the population gets a flu shot — just in the US alone! The virus looks like bit different each and every year, which is why we need to get a slightly different vaccine to protect against each strain.

One little virus causes so many people headaches, fevers, and more pain. But what if we could use viruses like the flu to help cure diseases instead of harming us by creating diseases? That’s where viruses come in as a form of gene therapy.

What exactly are viruses?

They’re microscopic parasites that need another cell to serve as a copy machine, and make more of themselves.

The structure of a virus is as follows:

  • The viral genome (either RNA or DNA) is surrounded by a protective coat of protein called a capsid.

In gene therapy, viruses serve as a carrier to deliver a new gene into the cell, which creates a mutation known as an insertion. The key thing is that viruses themselves are genetically modified so they don’t cause disease when injected into a patient.

The viral vector can be injected directly into a patient’s tissue, where it’s taken up by individual cells, or it can be introduced to cells taken from a patient’s body and put onto a cell culture, and are then re-introduced the cell.

What are some different types of viruses?

Retroviruses have genomes made of RNA.

  • Retroviruses directly integrate new genes into their host cell’s chromosome.

The icosehedral Adenoviruses are made of DNA, not RNA!

Adenoviruses allow for the transmission of genes to enter the nucleus, but not be integrated into the host cell’s chromosomes.This is actually an advantage of adenoviruses: no important host cell process, like mitosis (cell division), has a chance of being disrupted! In the case of cancer cells, this means that the already increased cell division won’t accelerate even more, thus spreading the cancer at an astronomical rate.

The structure of the DNA-based genome is key for generating a helper-independent virus:

(Vorburger et al)

Regions in E1, E3 and between E4 and the genome can either accept insertions or substitutions of DNA to help generate a helper-independent virus, which doesn’t need another virus to replicate.

Adenoviruses are found across all vertebrates, from fish to humans, but they show species-specificity! They’re separated into different sub-groups based on how reactive the antibodies they generate in the host cells are, analysis of their genes, and the overall gene content. Humans have about 57! For example, Subgroup A of the adenoviruses binds to a specific kind of receptor: the Coxsackievirus and adenovirus receptor (referred to as CXADR).

How we can use viruses to help treat cancer.

Cancer patients often have an immune response when they’re treated with cellular antigens, like those delivered in CAR-T cell therapy, or even with regular adenoviruses.

Dendritic cells are the key because they’re best equipped to present antigens and they can help activate the functions of T-cells.

What’s already being done?

The most common method is called Tumor-lysate pulsing, in which dendritic cells are collected from a patient’s blood and are then “fed” antigen proteins to be taken in to that cell.

What’s the point of using adenoviruses?

One of the key things about viruses is that they actually prefer to attack cancerous tissue rather than healthy tissues, according to LiveScience. But how do adenoviruses actually do this?

To answer that, I’ll ask you another question: what happens to a water balloon once you fill up with too much water? It bursts. And that’s exactly what happens to a tumor cell.

What happens when a virus enters a tumor vs. healthy tissue.

Once the virus enters the tumor through transduction, the virus replicates until the tumor cell bursts open like a balloon! When this happens, tumor cell proteins called antigens get released into the bloodstream. Antigens can be recognized by the immune system, and T-cells can rush over to and enter the tumor site, destroying what’s left of the tumor.

To sum it up, Dr. Antonio Chiocca, neurosurgeon-in-chief and chairman of the department of neurosurgery at Brigham and Women’s Hospital in Boston said it best. “The idea is very simply to place a viral infection in the tumor to alert the immune system — wake it up to the fact that there’s a tumor there.”

Beyond just naturally infecting and killing tumor cells, viruses can also be introduced into tumor cells to secrete compounds like Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) which accelerates the production of white blood cells to help fight tumor cells.

The E3/49K protein of subgroup D adenoviruses helps regulate immune response. When it is synthesized, it is then broken down and produces a compound that binds another protein called CD45, that suppresses T cells.

When a tumor cell is injected with an adenoviral vector, the fiber it secretes blocks the receptors of that virus that belongs to the surrounding cells. This may seem like a problem, but because the virus spreads slower, there’s less of a chance that the virus will go haywire and cause a different and more adverse immune response. This way, the virus and its host can work together in harmony!

How to generate an oncolytic virus:

So by now, you must be wondering “How do we actually generate an oncolytic virus?” In the words of Rhett and Link, “Let’s talk about that!”

The conventional method of generating oncolytic viruses is through homologous recombination, which is the process of exchanging nucleotide sequences between two similar or identical molecules of DNA.

There are three main methods of editing viral genome:

  1. Bacteria-based homologous recombination

All of these processes are extremely inefficient and time-consuming. We want to utilize the power of oncolytic virotherapy ASAP so we can save millions of lives!

That’s where CRISPR-Cas9 comes in as a relatively more efficient way of creating oncolytic viruses (read my article on how it works here!). As outlined briefly by this research, and using the Benchling software, I simulated the knockout of the thymidine kinase gene in the herpes-simplex 1 virus (HSV-1).

How I Did It:

I started by finding the genome of HSV-1 and locating the TK gene, which is the target DNA for this simulation. (Note: I’m indebted forever to the folks at GenBank for providing me access to the entire, fully annotated genome of HSV-1!)

This is just the first 250 base pairs of TK. As you can see, it’s a pretty long gene!

Next I needed to generate the PAM. The PAM is a short (2–6 base pair) sequence that tells the Cas protein where to start cutting. It’s usually found downstream of the target DNA and gRNA.

Each Cas protein has a specific PAM it works with. Cas-9, a nuclease derived from the bacteria S. pyogenes, recognizes the PAM 5'-NGG-3', where can be any nucelotide base.

The PAM is located few base pairs after the end of the TK gene, as marked by the pink arrow. The sequence is 5'-ACAAGG-3'.

A close up of the PAM sequence

Lastly, I designed my guideRNA (gRNA) which binds to the target DNA and in this case, induces loss of function in the TK gene. To bind to the target DNA, the gRNA has to be the complement.

The last 300 base pairs of the TKgene + DNA up to the PAM

And there you have it! The blueprints for using CRISPR-Cas9 to generate a modified oncolytic virus. The same research found that 50.1% of all the experimental viruses contained the mutations induced by CRISPR-Cas9, indicating its effectiveness!

As with all things, virotherapy has its issues. Let’s focus on two of the biggest ones: poor tumor targeting and the immune response.

Poor Tumor Targeting

In blood, adenoviruses interact with many biological compounds that often limit the amount of capsid available to reach tumors. The amount of liquid in blood removed by the kidneys has a half life of mere minutes, and when neutralizing antibodies induced by the introduction of viruses are thrown into the mix, that number is severely affected.

One way we could work around this, is by attaching polymers to the capsids of adenoviruses, or engineering cells as Trojan horses to deliver the virus to the tumor cells. This approach comes with its own issues, as a virus capsid, even if it’s genetically modified to do otherwise, will naturally induce neutralizing antibodies (as per the human immune response) and inhibit the rate at which tumor cells take in the virus.

To fully improve tumor targeting, the adenoviruses needs to be modified, using CRISPR-Cas9, to express a tumor specific compound known as a ligand. This ligand can be expressed as a protein on the surface of the capsid. The ligand and the tumor are like two opposite poles of a magnet — opposites attract!

Immune Response:

Oncolytic adenoviruses often provoke an immune response from a host. The cells synthesized from this response would theoretically help fight and destroy the tumor cells.

This also has to deal with the antigens produced by the tumor cells, as well as over-expression of antigens between the tumor and healthy host cells.

The most common thing would be to modify viruses to secrete compounds like GM-CSF (as mentioned earlier) to accelerate production of white blood cells.

Stimulating the immune system in this way presents itself with another problem: blockers like anti-CTLA-4 or anti-PD-1, which are immunomodulatory proteins, will not distinguish between viral and tumor epitopes (the part of the cell where the antibody attaches itself to). With the antibodies attaching to the wrong cell, the very thing we want to remain, the viruses, will go down rapidly!

To help prevent this, antibodies for anti-CTLA-4 or anti-PD-1 can be inserted into the host right before the virus, so that the tumor epitopes will dominate in presence over the viral ones.

Despite the potential issues using adenoviruses brings, it’s already shown clinical success!

  • T-VEC is an oncolytic adenovirus that works by infecting and killing tumor cells. It was approved as a drug by the FDA to treat melanoma, a form of skin cancer, in 2015.

Gene therapy is being used to combat the world’s deadliest diseases — who would’ve thought that viruses would be such an efficient way of delivering gene therapy to cells? As the saying goes, “one man’s trash is another man’s treasure”. Before you know it, you’ll be thanking a virus for helping you survive of the biggest diseases — cancer.

Key Takeaways:

  • Viruses are microscopic parasites that need other cells, called host cells, to reproduce and infect more cells.

Further Reading (Papers Referenced Throughout the Article):

Thanks for reading my article how viruses can be used to deliver gene therapy and treat cancer! If you’d like to connect and chat more about this topic, or anything specific to gene therapy and genetic engineering, I’m available on Linkedin, and can always be reached at my email: writetoapurva@gmail.com. Until next time!