The most common type of brain inflammation, viral encephalitis, is a global problem (Wang et al., 2022). A variety of viruses combined lead to nearly one and a half million new cases around the world every year. Between them, the herpes simplex virus is the deadliest and most commonly infectious in the western world, whereas the Japanese encephalitis virus is the largest cause of encephalitis globally. Descending from different species, the tick borne encephalitis and rabies add to the global encephalitis deaths as well.
cases globally per year(2019)
deaths globally per year(2019)
(Wang et al., 2022)
It is difficult to treat varying types of viral encephalitis, since both the cause and epidemiology of the infections differ greatly according to location, climate, and disease vector (Wang et al., 2022). Another hefty hurdle to get over in treating viral encephalitis is getting the therapeutics across the blood brain barrier to the infected neuronal cells (Teleanu et al., 2022).
We decided to tackle these problems by developing a novel therapeutic platform technology using siRNAs and liposomes to treat different varieties of encephalitides. Our focus in the project is the herpes simplex encephalitis as a model infection to possibly treat with a therapeutic created with this platform, as it is one of the deadliest viral encephalitides and currently has no vaccines against it.
The blood brain barrier protects the brain against toxins and pathogens circulating the blood, while allowing the necessary nutrients to pass through in order to reach the brain (Teleanu et al., 2022). It is a network of blood vessels and tissue made up of tightly spaced cells.
The permeability of the BBB is affected by many different cell types surrounding and constructing it, however, as the barrier fundamentally consists of lipid membranes of closely spaced cells, only water, certain gases such as oxygen, and lipid-soluble substances can diffuse across the barrier (Teleanu et al., 2022). Other substances such as glucose can be actively transported through the membranes. Therefore, most therapeutics don’t get to enter the brain through blood circulation, making it hard to treat encephalitis.
To address the aforementioned great difficulties in passing the blood brain barrier, our project introduces the nose-to-brain route as a solution. We plan to deliver our therapeutic as a liposomal aerosol created by an ultrasonic nebuliser, which can be applied to anyone to effectively treat viral encephalitis.
Our therapeutic has to have the ability to be taken up by both the olfactory and the trigeminal neurons in the nose, and get dispatched through the network of neurons across the whole brain (Lochhead et al., 2015). In order for an active ingredient to travel through the nose to the brain and work desirably, it needs to be protected and transported freely along neurons. For this purpose, we decided to use a fitting drug delivery system, which fulfils exactly this criteria: liposomes.
Liposomes are small vesicles with a diameter of 10 to 1000 nm, made out of spherical lipid bilayers with aqueous solution inside (Akbarzadeh et al., 2013). They have the same structure as the internal vesicles of the cells of almost all organisms, and they can easily fuse with plasma membranes of cells, which gives them the ability to release the contents of their aqueous cavity into the cell. This makes liposomes an effective carrier for many different therapeutics, all the while protecting their cargo from environmental hazards such as hydrolases, and offering a great biocompatibility.
Furthermore, special lipids or other molecules specific to certain targets on plasma membranes can be added to the surface of the liposomes, making the drug delivery a lot more efficient and decreasing the amount of possible side effects (Nogueira et al., 2015). At the moment, liposomes are gaining more and more popularity for drug delivery (Kraft et al., 2014), with prominent examples including the newly developed different mRNA-based COVID-19 vaccines.
It has been recently discovered that liposomes are transported retrogradely along the surface of neurons, meaning that liposomes administered into the nose cavity can attach to neurons of the olfactory area and travel all the way to the brain or to other ganglia (Lochhead et al., 2015). They release their content once they reach their target cells, where the carried drug will then take effect. This makes liposomes an effective way to deliver drugs against different viral encephalitides.
When developing drugs which should act in one of the most important organs of the human body, the brain, one needs to be especially careful with the drug design. In order to avoid any unwanted side effects which may cause huge damage to the person receiving the drug, it has to reach its target and act upon it as specifically as possible. Moreover, to not cause any inflammation or other harmful effects, it has to have a high biocompatibility. Also, since a big portion of encephalitis patients are immunocompromised (Saylor et al., 2015), this therapeutic needs to be fit to use in such patients. Considering all these aspects, we believe we have found the ideal type of compound as a drug against viruses infecting the central nervous system: siRNA.
siRNA molecules are short, double-stranded RNAs (dsRNA) consisting of 21 nucleotides (Elbashir et al., 2001). They are usually generated as a part of a eukaryotic cell’s defence against viruses: An intracellular helicase, the dicer protein, recognizes and cuts viral dsRNA into small fragments we call siRNA. Next, another protein, Ago2, binds to the siRNA and uses it as a template to identify and destroy a matching viral RNA (Ameres et al., 2007). This mechanism can be hijacked and used as a way of treatment: in this case, siRNAs are specifically designed to contain a sequence matching the mRNAs of certain viral proteins. With these siRNA molecules’ guidance, the Ago2 protein binds and destroys the matching viral mRNA Therefore, it inhibits the production of crucial viral proteins and a full assembly of viral particles. Thus, be it a DNA or an RNA virus, the siRNA keeps the infection at bay (Kaczmarek et al., 2017).
This way of treatment offers three great benefits (Elbashir et al., 2001): firstly, siRNA poses a very specific and effective way of treatment, as it only targets molecules with a matching sequence . Secondly, the siRNA is a molecule consisting of parts which naturally occur in all human cells, meaning that it has an outstanding biocompatibility and biodegradability as a drug . Lastly, once an optimal delivery method of the siRNA to its intended tissue is developed, one can target a plethora of different viral diseases in that tissue. This means, in the case of encephalitides, one can modify the drug to fit the encephalitis type by simply changing the sequence of the siRNA to the respective virus needing to be treated. This makes siRNA an outstanding platform technology with a high potential of being able to treat many different diseases in a comparably short time of treatment development.
In our project, we built a plasmid to produce our siRNA in E. coli (Huang & Lieberman, 2013). Aside from being a very affordable way of producing such a pharmaceutic, this method is also open to upscaling in the amount of siRNA being produced. Additionally, we determined the optimal lipid composition and production method for our siRNA-filled liposomes, both experimentally and through molecular dynamics simulations. We achieved this first by using the asymmetric centrifugation method (Massing et al., 2008), and in the future, we would utilise a microfluidics system to speed up and optimise the process of liposome production.
Finding the best proportions between the different lipids in liposomes can be quite tedious. Often, no direct proportionality is observed between the different ratios of components making up a liposome and certain properties of the resulting liposomes. For example, one can not oresee the size of the end liposome according to the amount of a certain lipid it contains. This means that researchers would have to try out every imaginable combination of different lipid ratios manually to determine the optimal composition of their desired liposome.
In order to make this process a lot easier and quicker for both our team and the whole field of pharmaceutical research, we came up with a concept for a semi-automated microfluidic system (Evers et al., 2018). This device would be used to test various lipid compositions quickly in succession. In this system, the lipid and the siRNA solutions will be injected into an interchangeable channel. As they flow along this channel, they are exposed to strong shear forces. This leads to the lipids naturally forming liposomes, with the siRNA trapped inside. Afterwards, the generated liposomes flow into a cavity, where their important characteristics such as their size are measured. Thereby, an assessment of each created lipid composition could be made very quickly, speeding up the process of research to match the current growing need of effective drug delivery systems into the brain.
Using our design, we created a novel pharmaceutical against herpes simplex encephalitis: Hersiran, a drug consisting of an siRNA mixture which targets the mRNA of vital proteins of the herpes simplex virus (HSV), packed in optimal liposomes to reach the infected areas in the brain across the BBB. Inflammation of the brain is one of the later stages of an HSV infection, which means this therapeutic could also be applied as a preventive method in the early stages of a detected HSV infection, protecting the brain from potential inflammation.
The herpes simplex encephalitis (HSE) has the highest mortality rates out of all viral encephalitides and, unlike the other types, there are currently no approved vaccines available for HSE (Kumar & Mendez, 2022). The most common and standard treatment to date is acyclovir, a drug consisting of a synthetic nucleoside analogue, and the current treatment of an encephalitis comprises three intravenous injections per day of the pharmaceutic, continuing for three weeks (Kumar & Mendez, 2022). This can greatly reduce quality of life and decrease the chance of people continuing to undergo this therapy. Furthermore, via the current therapy, the drug reaches the infection site in inadequate amounts to properly treat HSE (Kumar & Mendez, 2022), and it is also nephrotoxic, often leading to acute renal failure in patients (Yildiz et al., 2013). Another downside of acyclovir is the fact that in immunocompromised patients, the development of a resistance against this drug is a lot likelier (Piret & Boivin, 2011), which makes this already vulnerable group even more exposed to the fatal risks of HSE. This is why we created Hersiran, a safer and more effective pharmaceutical alternative, which can reach the infection site directly due to its local application.
In conclusion, by using siRNAs packed in liposomes to be delivered through the nose to the brain, we have created a therapeutic platform with an easy to produce technology to develop the necessary drug solutions for viral encephalitis varieties. With our system, the specific siRNA designed to fight against the specific viral encephalitis can be easily produced in E. coli and packed into the corresponding optimal formulation of liposomes, calculated with our microfluidics device. Therefore, not only can we transport therapeutics easier to the brain while circumventing the blood brain barrier, but we also have a better platform to develop more therapeutics for varying global cases of encephalitis.
The first step of improving our pharmaceutical would be making further different modifications of our formulation which would go beyond the scope of an iGEM project. For example, by using solid phase synthesis of siRNA with different chemical modifications, one could achieve more favourable properties such as a longer half life or a reduced immunostimulation (Deleavey et al., 2009). pH-sensitive lipids (Cox et al., 2022) and neuron-targeting surface modifications like Rabies Virus Glycoprotein and transferrin-conjugated liposomes as done in (dos Santos Rodrigues et al., 2020) would also be possible modifications to evolve our project even more. By building, testing and improving our self-designed microfluidics device, we could moreover reduce the time of finding the best ratio between the different lipid components drastically.
Our therapeutic would ideally be applied to the nose through tubes introduced into each nostril. This way, an aerosol containing our drug would be sprayed into the nose by a nebuliser. By developing a different application method, such as a gel patch for skin infections, even more tissues that are affected by the herpes simplex virus can be treated.
In order to further assess the safety of our pharmaceutical, more in vitro experiments should be performed. A cytotoxicity assay, and testing our drug in an epithelial as well as a neuronal cell culture are absolutely necessary steps to take in the future. Afterwards, first in vivo studies would follow, including a dose response study and a dose toxicity study. Once all these steps are completed successfully, the first clinical studies could commence, hopefully leading to an approved pharmaceutical which would help treat many people with lethal viral neuroinfections.