Therapeutic Nucleic Acids
Introduction
Nucleotides are considered one of the main components for life as we know it. They can act as signals, provide energy to keep your muscles going, and much much more. However, the most important role of nucleotides is to act as the code upon which life, in all its forms, is constructed. It is this central role of nucleic acids that could inspire a new wave of therapeutics, against conditions as varied as blindness and viral infections, all based on targeting the key to life itself.
Nucleic Acids
So what is genetic information and why is it so important? Well let’s look at the most widely known nucleic acid, which you may know as DNA. Deoxyribonucleic acid (DNA) codes for the instructions on how to build proteins. In turn, it is these proteins that carry out all of the processes you need to live. Fighting disease, breaking down nutrients, building new cells, all these things are done by proteins - the instructions for which are stored in DNA. The transfer of information in DNA to proteins is known as the central dogma. This involves the transcription of DNA into a similar nucleotide known as ribonucleic acid (RNA), followed by the translation of the RNA into proteins. Each segment of DNA that codes for a specific protein is known as a gene.
Figure 1: Diagram showing the Central Dogma. DNA is transcribed into mRNA, which is translated into protein.
DNA has one main function, the storage of information in the form of genes. On the other hand, RNA has a bigger range of functions, where each function corresponds to different types of RNA. The main type of RNA, which we will be covering later on, is messenger RNA (mRNA). mRNA is the type of RNA that carries the information from DNA in order to form proteins.
The structure of a nucleotide can be split into three main parts: a phosphate backbone, a pentose sugar, and a nitrogenous base. Depending on whether the nucleotide is a DNA or RNA, there are a few differences within the structure. DNA has a deoxyribose pentose sugar and the following nitrogenous bases: adenine, thymine, guanine, cytosine. RNA has a ribose pentose sugar and the following nitrogenous bases: adenine, uracil, guanine, cytosine.
Figure 2: Diagram showing the three main parts of a nucleotide, depending on whether the nucleotide is a DNA nucleotide (left) or an RNA nucleotide (right).
Figure 3: Diagram comparing and contrasting DNA (left) with RNA (right).
Now how are separate nucleotides linked to form a long chain of nucleotides? This is done through a specific bond called a phosphodiester bond. This links the phosphate backbone of one nucleotide to another.
Figure 4: Diagram showing two nucleotides bonded together via a phosphodiester bond.
Nowadays, the majority of drugs available function by interacting with different proteins that are found to be the root of the disease. However, most of the time, these drugs come with side effects. This is due to the fact that they interact with non-target proteins too (Nucleic Acid-Based Drugs｜SUMITOMO CHEMICAL, n.d.). Therapeutic nucleic acids (TNAs) are nucleic acids or related compounds that are used to treat disease (Martínez et al., 2015). They are a more recent type of therapeutics used in treating unmet medical needs. These therapeutics are able to target a disease at the genetic level by blocking the expression of disease-causing proteins.
TNAs have a high specificity, high functional diversity, and limited toxicity. Therefore, they show a lot of potential for the treatment of different diseases, infections, and even cancers. There are different types of therapeutics that TNAs can carry out. We will mainly be focusing on RNA interference (RNAi), gene therapy, and vaccines.
The most basic usage of nucleic acids is the inhibition of gene expression (Wraight & White, 2001). This means that any abnormal proteins that cause disease are stopped without affecting other proteins (Martínez et al., 2015). This will be explained further below.
Currently, most of the commercial drugs are those which contain small molecules that target proteins such as receptors or enzymes. However our bodies produce lots of very similar proteins the drugs would also end up targeting, resulting in side effects. These can be so serious that a specific protein cannot be targeted by simple drugs, which is the case for most of our proteins. On the other hand, pre-mRNA and RNA are targeted by the macromolecules known as oligonucleotides. Due to the fact that mRNAs carry the instructions to make proteins, oligonucleotides might be an effective type of therapy to treat diseases which are currently found to be untreatable by current drugs such as the genetic disease known as Duchenne muscular dystrophy (DMD).
The different nucleic acids mentioned above can be used in multiple different ways in therapeutics. For this page, we will be focusing specifically on RNA interference, gene therapy, and vaccines.
RNA Interference - Short Interfering RNA:
Let’s start off with RNA interference (RNAi). This can be carried out by certain types of RNA and DNA. The most common ones are short interfering RNA (siRNA) and antisense oligonucleotides (ASOs) (Creative Biolabs, 2022). These DNA or RNA molecules will bind to single-stranded nucleic acids whose base pairs are complementary. These single stranded nucleic acids are typically messenger RNA (mRNA) (Zhu et al., 2022). For starters, we will first dive into how siRNA works, before focusing on ASOs.
siRNAs carry out RNAi as follows:
1. siRNA is produced through the cleavage of double-stranded RNA via Dicer, which is an enzyme produced by humans.
2. When siRNA enters the cell, it is still double stranded. It will form complexes with proteins found in the cell, known as RNA-induced silencing complex (RISC).
3. The formation of the RISC allows the double-stranded siRNA to unwind, resulting in single-stranded siRNA.
4. The strand of siRNA that is complementary to the mRNA of interest will remain a part of the RISC and will look for said mRNA of interest.
5. Once the siRNA finds its complementary mRNA strand, it will bind to it. The enzyme component of RISC will cleave the mRNA.
6. The cleaved mRNA will be unrecognisable and perceived as abnormal to the host cell, and thus will be degraded, preventing translation.
(Davey, 2021)
Figure 5: Diagram showing the mechanism of RNAi by siRNAs.
But what disease exactly could siRNA help with the treatment of? Let’s look at a specific case study on the treatment of age-related macular degeneration (AMD). AMD is the main cause of severe visual impairment in people above the age of 65. When AMD is in its early stages, it is referred to as dry AMD. When it is in its late stages, it is referred to as wet AMD. Bevasiranib is an siRNA based therapeutic that is being analysed for its potential in treating wet AMD. What the siRNA in bevasiranib does is target a specific protein known as vascular endothelial growth factor (VEGF). This growth factor is involved in the process of choroidal neo-vascularisation (CNV) (Garba & Mousa, 2010). CNV is the formation of blood vessels in the region in front of the retina, known as the choroid. This blocks light from entering the retina. Hence, this phenomenon is what leads to wet AMD (Kovach et al., 2012). The siRNA prevents the protein VEGF from forming, so CNV does not occur, and wet AMD is treated (Garba & Mousa, 2010).
Now it’s time to focus on some challenges related to siRNA as a mode of RNAi. For starters, it could be the case that human cells notice the double stranded siRNA, mistake it as something a virus has produced, and end up destroying it. Obviously, once destroyed, it cannot carry out its function. Another problem that can pop up is that the cleaving of the mRNA (step 5 in the RNAi process above) does not occur, as the siRNA and mRNA are not fully complementary due to some mismatches in the bases. However, in spite of these challenges, researchers are working harder in order to figure out how these challenges can be overcome in order to use siRNA in RNAi more effectively (Davey, 2021).
RNA Interference - Antisense Oligonucleotides:
As mentioned earlier, ASOs can also cause RNAi. Now, unlike siRNAs, ASOs are a type of DNA, not RNA. ASOs function quite similarly to siRNAs in terms of RNAi, so let’s dive right into their challenges and how they are overcome. The main risk is that ASOs become degraded by the cell before they can carry out their job of RNAi, so ASOs are generally modified in order to prevent this. So instead of the normal sugar phosphate backbone bonded together by phosphodiester bonds, the backbone is altered and made up of phosphorothioate residues (Kole et al., 2012). This is the same as the original backbone, except the oxygen atoms that do not participate in the phosphodiester bonds are replaced by sulfur atoms instead (Sridharan & Gogtay, 2016).
Figure 6: Diagram highlighting the differences between a normal, unmodified backbone of a nucleic acid (left) and a modified phosphorothioate backbone of an antisense oligonucleotide (right).
The first ASO that passed clinical trials was made into a drug known as fomivirsen, which started being distributed and sold in 1998. This drug is taken when the person has retinitis, specifically retinitis caused by the viral infection of the cytomegalovirus (CMV). The ASO present in fomivirsen is complementary to the portion of mRNA of the virus that is involved in viral replication. Hence, the drug prevents the virus from replicating, which stops the virus from causing more harm (Sridharan & Gogtay, 2016).
Gene Therapy:
The next type of therapeutics that we will be diving into is gene therapy. Gene therapy is another form of therapeutics that involves nucleic acids. You can think of gene therapy as a sort of means to “correct” for genes that are causing some kind of hereditary and/or genetic disorder. And this is usually due to the fact that said genes are defective in some way (Misra, 2013). Gene therapy can occur in four main different ways:
1. The first way is by adding a normal version of the defective gene into a random part of the genome. The normal gene would replace the defective gene. This is the most common way of carrying out gene therapy.
2. Another way is by switching out the defective gene with a normal gene.
3. One alternative way is by repairing the gene itself, through a process known as selective reverse mutation.
4. Last but not least, gene therapy can also be carried out by modifying a specific part of a gene that is in charge of regulating itself, which means controlling the extent by which the gene is turned on or off.
(Misra, 2013)
Figure 7: Diagram highlighting the different types of gene therapy, as explained in the list above.
RNA Vaccines:
Now that we have a good grasp on what nucleotides are and how they can be used for therapeutic reasons, let’s put this all into practice. Specifically, let’s look at a clear example of a therapeutic nucleic acid you likely heard of recently: mRNA vaccines. This was the main type of vaccine used to combat the infamous COVID-19 pandemic, and launched the topic of nucleotides as therapeutics into public discourse. To delve into this topic, we need to first discuss the basics of our immune system. Specifically, we are looking at the adaptive immune system, which is the most effective component of our immune system. It is capable of rapidly removing any pathogens, including those hiding out in your own cells. However, for the adaptive immune system to actually work, it has to have already come into contact with the pathogen beforehand. Remember, pathogens, like us, also rely on proteins to function, but use proteins that are different from our own. Thus, the adaptive immune system remembers the proteins expressed by past invaders to use them as targets when they invade again. mRNA vaccines exploit this principle by inducing your own cells to produce proteins that are normally produced by a virus, like SARS-COV-2. The immune system detects these foreign proteins and records them in order to mount an effective response in case the pathogen is actually encountered. Therefore, the mRNA vaccine makes your cells produce parts of pathogens so your immune system knows what to look for and target in case of a real infection (Linares-Fernández et al., 2020).
However, why use an mRNA vaccine specifically? After all, previous vaccines used the proteins or even the dead virus itself, so why not do that with COVID-19? Well the reason is time and efficiency. It takes a lot of time and resources to grow a pathogen in large enough quantities to be able to commercialise its components as a vaccine. Also, keep in mind that this process has to be done for every single different pathogen you want to vaccinate against. Compare that to mRNA vaccines, where any lab can make the vaccine as long as they know the sequence of whatever protein they are producing. Additionally, the vaccine can be kept largely the same, as all that needs to be changed is the particular sequence of the mRNA. This means that mRNA vaccine technology allows us to respond to new infections much quicker and with less effort than traditional vaccines (Xu et al., 2020).
This explanation, however, raises a question of its own. If mRNA is so much more cost-effective than traditional vaccinations, why is it only now that we are hearing about such vaccines? Whilst it’s true that these vaccines have a lot of potential, there were a number of roadblocks to getting such technology to this point. The main issue is that, normally, RNA found outside a cell is a signal to the immune system that something has gone very wrong, and so it mounts a very aggressive response that could, at worst, put the patient into septic shock and death. Additionally, RNA is a very unstable molecule, which in most situations is a good thing because it means that cells can readily alter which mRNAs are present in order to change their behaviour. However, as you can imagine, instability is not something you want in medication. Whilst this is all true, it should go without mentioning that the vaccine was stable enough to be administered to patients without immediately triggering extreme reactions. This has mainly to do with advances in our understanding of both chemistry and RNA modifications. Our cells tend to place lots of small chemicals on mRNA, effectively decorating it so that it does not tend to provoke immune responses or be degraded too quickly. Developing both an understanding of these decorations, along with how they can be produced outside of cells, is what allowed vaccines, such as those for COVID-19, to be developed in the first place. Thus, the use of nucleotides for therapeutic applications has real tangible impacts on us all that will shift our response to both common diseases, as well as pandemics (Wang et al., 2021).
TNA Delivery - Viral Vectors:
One of the main hurdles for any treatment relying on nucleic acids is the delivery of the nucleic acid into the cell. One example is the delivery of the nucleic acid via a viral vector. A viral vector is simply a virus that has had its own nucleic acid replaced by the nucleic acid of interest to be inserted. This nucleic acid of interest could be an siRNA or an ASO for RNAi, or a gene for gene therapy. Depending on the type of virus, the nucleic acid of interest can enter the human cells and carry out its function. For example, some viruses will go about it by remaining outside the human cell, and inserting the nucleic acid into the cell. On the other hand, some viruses pretend to be protein molecules in order to be allowed access inside the human cell, and end up entering the cell whole before releasing the nucleic acid of interest (Misra, 2013).
Let us give you a concrete example. The first virus to be used in such a way was the retrovirus in gene therapy. It has been most successful in treating X-linked severe combined immunodeficiency (X-SCID), a severe genetic disorder that affects the immune system (Misra, 2013). It only affects males, resulting in constant and persistent infections, pneumonia, rashes, amongst many other problems (National Institutes of Health (NIH) & Genetic and Rare Diseases Information Centre (GARD), 2021). Now let us get back to the retrovirus, and how it carries out its job. The retrovirus enters the host cell, which is a human cell in this case. Once inside the cell, the virus’s enzyme called integrase will integrate the gene that was delivered by the virus into the host cell’s genome (Misra, 2013). And just like that, the retrovirus has acted as a vector in gene therapy.
Figure 8: Diagram demonstrating the mechanism of using a viral vector as a means of TNA delivery.
Like most things unfortunately, there are some limitations of using retroviruses as vectors. The main one is the fact that the integrase enzyme can sometimes add the gene from the virus into a random part of the genome. If the gene happens to be inserted in the centre of another gene, then this can result in harmful mutations in the DNA. If the gene is added into a gene that is in charge of cell division, then there will be an increase in cell division that is not properly regulated, which can lead to cancer. Fortunately, these problems are being worked upon, and some solutions have been found. One such solution is by including a sequence that shows the gene where to insert itself. An example of this type of sequence is the β-globin locus control region (Misra, 2013).
TNA Delivery - Non-Viral:
Now let’s say that you do not want to use a virus as a means of transport. The delivery of a nucleic acid can also be carried out via a non-viral delivery method. For example, this can be done through direct DNA injection. In fact, there were successful clinical trials that proved that this method of delivery works and can be used for the desired functions. Other methods of delivery exist, but the one that seems to be the most promising is receptor-mediated gene transfer (Misra, 2013).
The non-viral delivery method works, but still needs to be further improved upon. This includes finding a way to place the gene of interest inside the genome in a stable way, and improving how well the non-viral delivery capsule can carry the gene of interest (Misra, 2013).
Conclusion:
Using nucleic acids as therapeutics has resulted in new ways to go about treating diseases. Like most things, there is much room for improvement. However, therapeutic nucleic acids have proven to have a lot of potential in therapy, specifically through RNA interference, gene therapy, and as a vaccine.
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