Viruses Might Be Our Next Best Friends: Using Viruses as Vectors in Gene Therapy

This is the elusive Covid-19. It is highly infectious and targets cells present in the lungs.

Viruses have been at the forefront of media, as the Covid-19 virus continues to wreak havoc. However, can we use this destructive power of viruses and harness it in order to help humanity? Maybe! In different areas such as gene therapy, they are being used as vectors to deliver genes into cells. Ironically, they even may combat different diseases instead of causing them.

Viruses 101

Diagram of a virus. Source:https://biologydictionary.net/virus/

Shown above is a diagram of an enveloped virus. A genome is encapsulated inside a capsid(protein container), which is then hidden inside an envelope. This envelope is made out of an original host cell’s membrane, and has different proteins on it that allow for proper binding and attachment to another target cell.

Alternatively, viruses can also opt to not have an envelope and instead just have the capsid contain different proteins that will attach to cell receptors. Enveloped viruses are much easier to combat as the envelope can sustain environmental damage and once it lyses(breaks down), the virus loses its receptors and the naked capsid won’t be able to infect cells.

Viruses are also in a weird spot when it comes to classifying. Are they living? Or are they dead? They certainly don’t seem to be dead, but then again, they can’t even reproduce by themselves. Although I won’t be talking too much about this massive debate, it is definitely important to keep in mind, as this seemingly dead “thing” doesn’t look so dead after all.

Viruses can be spread in many ways, which is a bummer. They can either be transmitted through things such as bodily fluids or even through different vectors, such as mosquitoes and fleas. And, since it is easy for such viruses to infect us, we have many ways to combat and fight back.

Although there are sooo many different viruses, they are all essentially just genetic material inside of a protein box. However, whether they are RNA or DNA makes a big difference when it comes to how they infect and how they reproduce. I will go into more depth when I talk about how viruses reproduce.

There are many different types of viruses and there are a whole variety of shapes and sizes that they come in. They range from tiny geometrical shapes to large and bizarre shapes.

Bacteriophage

Source:http://textbookofbacteriology.net/phage.html

One example would be the bacteriophage. As shown above, it really doesn’t look like anything natural. It sort of looks like a mini rocketship, albeit a bit more creepy. These little guys are specialized into targeting specific bacteria(based on species), and they are really good at it. They kill almost forty percent of all bacteria that are in the oceans every single day. And due to this, they are being intensely studied as they have been shown to fight bacterial infections without massive drawbacks!

Adenoviruses

Source:https://web.stanford.edu/group/virus/adeno/2004takahashi/webpage/second.html

Shown here is an adenovirus and they look pretty nifty. They are shaped like an icosahedron, with fibers sticking out at each vertex. They are pretty common, and they can give you a wide range of symptoms, such as the common cold, fevers, pneumonia, acute gastroenteritis(inflammation of stomach or intestines), and even neurological diseases(extreme cases). However, these guys have been very helpful in gene therapy. Since they have a high transduction rate(transfer of genetic material), we have been inserting beneficial genes into these guys to help people who are suffering from different diseases such as lung cancer. However, since they do often stimulate an immune reaction, there is still more work that needs to be done.

Retroviruses

Picture of a lymphocyte(category of a white blood cell) that has been infected with HIV. The virions(shown in green) are budding out of the white blood cell. Source:https://en.wikipedia.org/wiki/HIV#/media/File:HIV-budding-Color.jpg

HIV is one of the most infamous viruses as it is one of the scariest viruses. It is part of the retrovirus family and does things a bit different than normal. Rather than just hijacking a cell’s machinery to do its bidding, a retrovirus has an enzyme called reverse transcriptase. This allows for the RNA of a virus to be made back into DNA. This viral DNA is then directly integrated into the host cell genome(called the provirus) and stays with the cell, and its daughter cells. This is dangerous as it essentially weakens the cell and also makes it impossible to get rid of it. Since HIV targets T-Helper cells(white blood cells) this cripples the power of the immune system drastically, making the infected person prone to other infections.

Stages of Viral Reproduction

In viruses, there are seven(more or less)general stages of reproduction. Being that viruses can’t reproduce on their own, they must access the machinery of other cells. This type of reproduction really blurs the line when it comes to the question of whether viruses are living or dead, but the way that they do this is absolutely incredible and frightening at the same time.

The first stage in viruses is to attach to the host cell’s membrane. Through adsorption, the virus essentially can merge and with a cell’s membrane.

After the virus attaches, the capsid will enter the cell. Enveloped viruses will shed their membrane, and the membrane will fuse with the host’s membrane, including all of its receptors and proteins. This leaves a capsid that still remains and enters the cell.

The capsid of the virus is then dissolved inside the cell. This can either be through the viruses own proteins and enzymes, or by the lysosomes of the host cell. After this happens, the virus’s genetic material and proteins are free to use the host cell’s machinery.

Here you see how different families of viruses replicate. You can see that they all will eventually synthesize mRNA as well. Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149704/bin/gr1.jpg

Once the capsid is lost, the genetic material is free to work its magic. There are many nuances to the ways that different viruses will replicate, but I will go over three different general cycles, based on their genetic material. Keep in mind, that this is a general overview, as even DNA viruses differ in many ways.

DNA

DNA viruses will have the DNA enter through the nuclear pores of the cell, so that they can access the nucleus. After this, viruses are free to use the host cell’s machinery to transcribe and replicate their DNA to create more virions.

Here you see how different RNA viruses replicate! Source: https://mmbr.asm.org/content/77/2/253/F7

Positive RNA

Positive RNA or plus-sense RNA can already act as mRNA. This means that they will be able to directly translate into proteins via ribosomes. After translation, early proteins are synthesized that will help with replicating more and more of the original template RNA.

Negative RNA

Negative RNA or minus-sense RNA cannot be translated into RNA, but acts as a complement to positive RNA. Negative RNA will have their own machinery and proteins that will first transcribe the negative RNA into its complementary strand and thus a positive RNA. This positive RNA then is read by the ribosome and manufactures proteins that can aid in replication or in other viral parts. It is important to also remember that different proteins will also have to be included in each new virion, as it needs to be transcribed into positive RNA before funky things start happening.

Retrovirus Style

As I stated before, retroviruses are special because they utilize a special enzyme called reverse transcriptase. Rather than performing the normal steps of RNA viruses, they will have their RNA turned into DNA. This is will then be integrated into the host’s genome, and it is called a provirus. Then, it will follow the normal steps in normal DNA transcription and translation and they will forge virus parts accordingly.

I sort of talked about this in the last step, but viral parts will be synthesized as the virus has its genome read and changed into mRNA. Viruses will first synthesize proteins that help with the replication process. Eventually, different viral proteins will all be produced, but they will have to be assembled together.

Viruses have their parts assembled and produced in the rough endoplasmic reticulum and the Golgi Apparatus. Assembly of the viral proteins and packaging of the capsids will occur, and once that happens, they will be ready for transport. Enveloped viruses will also have their new receptors produced and expressed on the outside of the host cell’s membrane.

Once virions are assembled correctly, they are clear for departure. Enveloped viruses will take some of the host cell’s membranes, and the newly produced receptors with them. Then the viruses are free to repeat this cycle over and over again.

This process that viruses go through, makes them seem a lot less robotic and lifeless, don’t you think? They aren’t exactly “living” as they don’t check all the marks like we do, but they definitely seem lifelike. Although these guys seem pretty scary, we still are able to survive these viral infections. Hell, every single day, you are exposed to millions of viral particles, but thanks to your immune system, they are stopped(most of the time).

But how can we use these guys for good?

Well, as you saw above, they are incredibly skilled at getting inside a cell and using its machinery! So, what scientists have done, is that they have just changed the genetic material inside, and thus make viruses useful!

Becoming Unlikely Friends

In the 1980s, we finally began to see success and promise in this field. Although there have been successes, there have also been many detrimental failures. Using viral vectors is a slippery slope, as once we try to do therapy in vivo(in the organism), we will have to have a diverse knowledge of specific receptors on different cells, to how the immune system will react.

We have come to use many viruses in gene therapy. The most common ones are retroviruses, adenoviruses, adeno-associated viruses(AAVs), naked DNA, and herpes simplex viruses. Shown above, you can see that adenoviruses and retroviruses are the top dogs when it comes to use in clinical trials. But how do these viral vectors differ from their wild-type counterparts and how do they work?

Retroviruses are special viruses since they actually integrate into a host cell’s genome. This is very effective when trying to treat diseases long term as they will stay with the patient for as long as those cells stay alive.

Synthesizing a retroviral vector! Source: https://www.genetherapynet.com/viral-vector/retroviruses.html

On top, you see a normal retrovirus which has its psi(the trident looking thing), gag, pol, and env proteins. The psi protein is the packaging protein, and that is important when you want to make actually functional viruses. The group specific antigen(gag) codes for proteins necessary to keep the structure of the virus, and other proteins essential to the proliferation of the virus. The polymerase(pol) gene codes for the elusive reverse transcriptase, and other proteases and integrases, which help with the integration of viral RNA into the host genome. Then, the envelope(env) codes for envelope proteins.

For therapeutic purposes, there is no point to using the gag, pol, and env genes, so we can replace them with a therapeutic gene, giving us around 6,000 to 8,000 base pairs that we can insert into the capsid.

But how can we transfer this modified genetic material?

Well, its pretty simple! We just take a cell and insert the gag, pol, and env genes into it and it will code for the proteins no problem. Then we just need to insert our new genome in, and it will just package them up, and we will have fresh vectors ready to go!

But there are still some issues…

Drawbacks

First and foremost, retroviruses must infect dividing cells, which is not the most common state that a cell will be in. Since these guys interact with the actual genome of a cell, there is also a chance for oncogene activation, which can lead to cancer developing. In a French clinical trial, three out of eleven patients who were being treated for Severe Combined Immunodeficiency Disease(SCID) had developed leukemia. This leads to the halting of further tests, but we have now understood the limitations and have deemed it rather safe to use retroviruses in further test trials.

Just injecting DNA works as a gene therapy treatment. Since we don’t need to have a limit on how much we can carry, or immune response, this is a pretty safe way to transfer DNA.

The main reason why we use viral vectors, is that there is a really low rate of expression through naked DNA. Moreover, we can’t use IV and we must inject it intramuscularly. This has led to some nifty ideas, like a gene gun. This covers the DNA with gold molecules and it shoots the DNA very quickly so that it can be integrated into cells more efficiently.

Liposomes have also been used as they have been seen to greatly increase the rate at which DNA can be integrated and expressed! Adding on to the fact that you can’t develop an immune resistance to DNA, you can inject DNA as many times as you want without worrying about efficiency rates going down.

Adeno-Associated viruses have become a very common viral vector. They are are part of the dependovirus family, as they require for another virus(adenovirus) to infect the same cell as they do in order to replicate. Although they are small and have a very small carrying capacity (around 5000 base pairs), they have many advantages, as they can avoid stimulating an immune response and also are not usually shut down by the innate immune response.

Production

Using cells to make AAVs. Source: https://www.nature.com/articles/d42473-018-00113-0#:~:text=Newer%20iterations%20of%20the%20helper,and%20package%20viral%20genetic%20material.

A newer type of AAV production is similar to retroviral reproduction, as it takes a cell expressing the rep and cap genes, and in conjunction with an adenovirus and hybrid, it can create AAV viruses(and some other helper viruses). By adding a transgene inside, we can actually take out the hybrid virus out of the original step, and thus create AAVs as well. These cells have been dubbed the packaging cell and producer cell, respectively.

There are also many other ways to create AAVs, but wanting to keep this shorter, I will be skipping them. However, it is important to keep in mind, that the main thing that they all have in common is that they cost a lot and that it it wouldn’t scale up very well.

Drawbacks

The main drawback is the small storage size. A measly 5,000 base pairs is still enough for some gene transfers, but it is not enough for others. However, there have been efforts made that actually combine two AAVs together, creating a genome with a capacity of 10,000. Another drawback is the high cost. It costs a lot to create these cells, and scaling would require scaling out rather than scaling up.

Now we are finishing off with probably the coolest and most complicated vector when it comes to preparation. Adenoviruses are very common as they are both transient(do not integrate into genome) and that they have a high storage capacity (around 25,000–30,000 base pairs). However, this is the latest generation, and there have been many iterations of this viral vector.

Some viral awesomeness! Source: https://www.nature.com/articles/3302612

This might be a scary looking graph for some of you, but don’t worry, we will get through this together! On the top, you see the Human Adenovirus Type 5(Ad5) and its normal genome. All those places that you see arrows and blocks are regions that code for the respective protein. All the proteins that start with E are the early coding proteins.

First Generation Vectors

In the first generation, the E1 and E3 coding regions are deleted as they spur viral reproduction, and that is definitely not what we want when it comes to a therapeutic treatment. However, there is still some gene expression that leads to the immune system cleaning up infected cells(killin em). This means that we have done this all in vain.

Second Generation Vectors

Second generation viruses have all of their E regions deleted, and this opens up around 14,000 base pairs for a transgene(transfer gene). However, their is still some immunotoxicity with residual viral genes being expressed.

Third Generation(Gutless) Vectors

This leads us to the third generation, also dubbed the gutless Adenovirus vector. This means that it is missing almost all of its viral parts, and it requires a helper virus to form it. Now, there is almost all of the adenoviral genome for the taking, leading to almost 37,000 base pairs that we can insert. However, since most of the genes we want to insert is not that long, we need buffer DNA. This is also a very hard process, as we need for DNA that won’t do anything wonky with our new gene or code for any new proteins. Now, I will explain to you the real meat of how to make a gutless adenoviral vector. Keep in mind that this is a very complex process, and I am missing out on a lot of parts to simplify the process, but here we go!

Developing a gutless adenovirus(with some impurity) Source: https://www.nature.com/articles/3302612

Alright, so above you, you see the insertion of the helper adenovirus and the gutless adenovirus genome into a special cell that has the Cre/293 recombinase(very important). The helper virus genome is a normal adenovirus with all of its viral coding parts. It then has its packaging protein(the trident thingy, also called psi)flanked by two loxP sites. The helper genome codes for proteins that help with DNA replication! The helper genome also encodes for viral capsid parts, and then they are ready to be packaged.

Now the Cre/293 recombinase(which I mentioned a bit earlier) looks out for two loxP sites, and if it sees it, it will cut out the part in between them, and join them together. This essentially gets rid of the helper genome’s capacity to get packaged back into the virus. This leads to the gutless genome stealing all of the hardwork of the other virus, and it is packaged into the capsid with all the appropriate proteins.

And voila! You have yourself a gutless vector. However, there are still some problems. One is that a small percent(0.1–10%) of the helper virus’s genomes actually get packaged into the capsids. This impurity is still a major problem, because we don’t want to inject people with live and dangerous viruses.

Drawbacks

Although it has so much poured into it, adenoviruses still have some glaring issues. The main issue being that they still elicit immune responses and can get people sick. Moreover, they target cells with the Coxsackie and Adenovirus Receptor(CAR). Although most tissue types display this receptor, the liver cells especially express this receptor, leading to added toxicity being possible. Also, the serotypes(specific viral species) that we use for gene therapy are Ad5 and Ad2, which are also very common viruses. A lot of people will already be immune to these adenoviruses, and that means that not everyone can be treated with these serotypes. Although proposals to use other serotypes have been proposed, we still need to learn more about them and the effects they have on the human body.

Helping Viruses Out

Viruses are great at infecting our cells, but we have also made many modifications so that they can better express the therapeutic genes put inside of them

The capsid or envelope’s receptors are the key way that a cell can infect cells. They have proteins that specifically bind to specific receptors. For example, the adenovirus binds to the CAR on cells. Although this provides specificity, there are many caveats.

If a virus was good at infecting most cells, then there would be a massive problem if we were to use the virus in vivo. The virus would essentially infect every cell, and since most use of this gene therapy is to target cancer cells, then this would be horrible news if we delivered a suicide gene to a perfectly normal and healthy cell.

On the flip side, if we want the virus to target a cancer cell, most viruses don’t display receptors that specifically target cancer cells, because that would not be beneficial. Sometimes, we need to get viruses to be able to target specific cells, and we can do that by modifying the proteins that are present on the virus.

Although modifying the capsid would lead to problems such as assembly, creating chimeras, or mixtures of new and old proteins can lead to a new creation. For example, Adenoviruses have a fiber that binds to the CAR receptor. By adding ligands(special proteins) that will bind to other specific receptors, the adenovirus vectors can now target cells with different types of receptors!

Conclusion

Like every type of science, we need to tread lightly and cautiously when experimenting with new technology. There is so much that we don’t know about viruses. And yet they have so much potential. We need to be respectful of their power, while also being appreciative and scientific with their use. As shown by using nuclear reactors, even one minor failure can set back the industry decades in progress. Gene therapy has a lot of promise in the coming years, but we need to be careful because viruses can be both our friends and our enemies.

Thank you for reading this article! I’m Sam and have a real passion and hunger to learn! I am really interested in biotech and space tech. You can check out my other articles, which are based on technology and a little bit about mindsets. If you want to contact me, you can connect with me on LinkedIn, or email me at samlitks2@gmail.com! Thanks again and have a wonderful rest of your day!

Just trying to make sense out of all there is to know