Our aim was to develop an siRNA platform technology against viral neuroinfections. Herpes simplex virus (HSV) was chosen as a demonstration target. The target protein VP5 is expressed by both HSV1 and 2, thus making our pharmaceutical potentially effective against both types. Especially HSV-induced and possibly lethal encephalitis, most often occurring in new-born children infected during birth, in immune-suppressed patients during cancer therapy, or after organ transplantation, was chosen as indication.
Synthesis of a specific siRNA able to efficiently prevent the expression of the viral protein in cell culture has been proven. This biotechnological approach poses a more environmentally friendly way and reduces the usage of problematic solvents and chemicals compared to classic solid-phase synthesis. Synthesis in bacteria also results in production of several siRNAs against the target, as the precursor molecule is cut into several effective siRNAs. Therefore, the development of resistances against therapeutics of this platform is likely to be drastically reduced.
To overcome some innate problems of siRNA, such as low stability, poor pharmacokinetics and off-target effects, a system of liposomes was chosen as a carrier. Liposomes enable the circumvention of the blood-brain barrier via the transport along neurons originating in the brain and ending in the nose. Furthermore, this protective hull prevents degradation of the active pharmaceutical ingredient (API) as RNases would quickly degrade any applied RNA inhibiting the accumulation of a therapeutic dose (Hu et al., 2020). This especially applies to the nasal application as its mucus is filled with enzymes destined to fight off pathogens. Furthermore, free RNA triggers a reaction of the immune system by activation of toll-like receptors and RIG-I-like receptors. Cytokines and interferons are released and can possibly lead to inflammation and other adverse effects (Tatematsu et al., 2018).
To improve the tolerability of our pharmaceutical we would exchange the cationic lipids with ionisable lipids. Cationic lipids are known cytotoxic agents, but greatly increase the uptake of negatively charged siRNA and the endosomal escape of the liposomes. Ionizable lipids are neutral at physiological conditions, thus minimizing their toxicity. They can be protonated at acidic conditions. Thus, it is possible to produce the liposomes at acidic conditions so that the positively charged lipids increase RNA intake. Endosomes contain an acidic environment also resulting in the protonation of the lipids which is known to increase their endosomal escape. This prevents the degradation of the carriers and their cargo. Especially the use of unsaturated or multi-tail ionizable lipids have shown increased cargo delivery (Han et al., 2021).
To increase the uptake and penetration we would evaluate the addition of edge activators, e.g., Tween 80, to our liposomes which results in more deformable and flexible liposomes called transferosomes enabling the carriers to pass pores that are even smaller than themselves. As this also destabilizes the liposomes the trade-off between stability and penetration would have to be closely evaluated (Rajan et al., 2011).
Nasally applied liposomes partly circumvent the blood brain barrier by passing through the olfactory or trigeminal pathway (Hong et al., 2019). Therefore, the proposed implementation is an ultrasound based liposomal aerosol applicator introduced as two sondes into each nostril. This assures a precise and stable dose application. By additionally preventing nasal breathing with a clamp, the dose inhaled and thus entering the systematic bloodstream or the intestines resulting in a loss of efficacy is reduced. By coating the liposomes with chitosan their persistence against the mucosa is increased resulting in a higher uptake (Filipović-Grcić et al., 2001). The particle size of aerosols plays an important factor on their target location and efficacy. Small particles may reach the bronchi and bronchioles whereas larger particles > 10 µm precipitate in the oropharyngeal region and above. As the entrance of liposomes into the pulmonary causes systematic absorbance and is therefore unwanted, a particle size of more than 10 µm is desired. Droplets precipitating at the oropharyngeal region are swallowed and degraded in the digestive tract (Labiris & Dolovich, 2003). As only small amounts of aerosol would be released at a time, the majority would precipitate just after the sonde and therefore be available for uptake through the olfactory or trigeminal pathway. No first-pass effect and glomerular elimination take place, thus reducing the necessary dose. For achieving optimal settings for the nebuliser it is necessary to experiment with some factors. A decrease of vibration frequency and increase of ventilation results in larger droplets which are needed for this application. High vibrational intensity ensures efficient performance and a rapid nebulisation time (Flament et al., 2001).
As patients suffering from herpes induced encephalitis are already treated at intensive care units and are likely unconscious, the introduction of said device would not cause a problem. The stability of the liposomal formulation treated with ultrasound would have to be evaluated especially if the progression to transfersomes is carried out.
One issue of the nasal route is that the conditions of the nose, e.g., pH, tonicity and mucus composition vary significantly especially during rhinitis which might influence the uptake (Hong et al., 2019). As liposomal based siRNA drugs are usually well tolerated, the large therapeutic window should enable the usage of a higher dose to compensate for the reduced uptake into neurons. This would have to be confirmed in toxicological studies.
To prevent irritation of the nasal mucosa the finished product must be isotonic. As the mucosa exhibits little room for pH compensation the formulation is also supposed to be euhydric with a pH of 6.8 to 8.3. As citrate and borate buffers aren’t well tolerated for nasal application a phosphate buffer would be used (Fahr, 2021).
After successful testing and tweaking of our nose-to-brain liposomal siRNA platform, it is easily possible to synthesize siRNAs combating other neurological pathogens like Epstein-Barr virus, a known oncovirus and likely a risk factor for many other diseases like multiple sclerosis (Cao et al., 2021). Tick-borne encephalitis virus (TBEV) is another potential target. To this date no causal therapy for the life-threatening tick-borne encephalitis exists which mostly affects the eurasien continent. As death or serious neurological damage are common, medication would be a welcome gift (Bogovic & Strle, 2015).
Despite focusing on the therapy for HSV as an example, the real value of this project is the implementation of a platform easily adjustable to different neurological pathogens. At this point, treatment of most neurological infections is still complicated or impossible but this implementation might change this tragic fact to the better.
Another possible implementation is the topical application of siRNA carrying liposomes to treat dermal herpes simplex. Liposomes are able to penetrate the stratum corneum and rest of the dermis due to their interaction with lipids of the skin. They deliver their payload directly into the cells where the API is needed and therefore reduce the systemic absorption. Furthermore, they exhibit a depot effect which helps to sustain a steady concentration of the API (Pierre & Dos Santos Miranda Costa, 2011). As the skin around herpes blisters is damaged, the use of penetration enhancers is likely unnecessary as the liposomes should be able to readily reach the lower levels of the skin. By incorporating the liposomes into a gel patch known from treatment of cold sores, their advantages of providing optimal conditions for healing, prevention of viral spreading and fixation of the API are incorporated in our product (Karlsmark et al., 2011). It has been shown for finasteride that incorporation of liposomes in gels is a possible topical delivery system (Khan et al., 2018). This would also enable us to add dexpanthenol which is known to accelerate wound healing.
To achieve gelation, cellulose based gelling agents like methylcellulose are suitable. Unlike polyacrylic acid gels they are uncharged and therefore ionic interactions with the cationic lipids are avoided. The gelling agent would be added after the incorporation of the API into the liposomes (Madan et al., 2018).
Even with acyclovir treatment about 30 % of patients suffering from HSV induced encephalitis either die or receive a severe neurological deficit. Most of the other 70 % suffer from neurological symptoms as well (McGrath et al., 1997). Therefore, a dire need for a better treatment of HSV induced encephalitis exists. With our target-directed, high potency liposomal siRNA approach we hope to deliver this enhanced therapeutic.
Unlike acyclovir which is the standard therapeutic against HSV our innovation does not have to be applied i.v., which drastically reduces systematic side effects. To treat encephalitis the blood brain barrier requires the usage of a much higher concentration of the known nephrotoxic acyclovir. The nose-to-brain approach renders this unnecessary.
As acyclovir might be incorporated into cellular DNA it is a potential teratogen making its use during pregnancy possibly problematic. While current research does not suggest major teratogenicity (Mills & Carter, 2010), our product does not show any plausible reasons for mutagenicity. Yet, this would have to be tested prior to release.
Furthermore, resistances against acyclovir have been reported although only accounting to 0.5 % in immunocompetent and up to 10 % in immunocompromised cells of examined virus isolates (Bergmann et al., 2017). Our product is developed with immunocompromised patients in mind and is not prone to develop resistances due to the production of several different siRNAs as explained previously. Because acyclovir is used frequently and hardly any alternatives exist except for foscarnet which causes severe side effects and famciclovir and vidarabine which are only effective against HSV-1 (Mutschler, 2013), a novel therapeutic is a welcome innovation.
Furthermore, acyclovir must be administered up to five times a day (Mutschler, 2013) as it is only effective when a necessary serum level is available. As this siRNA-based drug causes gene silencing it is still effective for a certain time after administration and would therefore avoid the rigid therapy plan of acyclovir.
As with all therapeutics for human use approval by responsible authorities, e. g. European Medicines Agency (EMA) for Europe, the Food and Drug Administration (FDA) for the US and the Pharmaceuticals and Medical Devices Agency (PMDA) for Japan, are needed. In cooperation with those three agencies, the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) releases guidelines for pharmaceutical quality, safety, efficacy, and multidisciplinary. For clinical and preclinical studies that are needed prior to approval, the safety and efficacy guidelines provide counsel (Becker et al., 2017).
After successfully conducting the preclinical studies mentioned in the integrated human practices wiki and receiving approval, it is finally time for the first-in-human studies. The clinical stage is thus entered (Becker et al., 2017). Figure 1 depicts a graphic of this process.
It is optionally possible to conduct a phase 0 study. This stage involves fewer than 20 people who are administered subtherapeutic doses to gain further knowledge about pharmacokinetics and -dynamics (Kummar et al., 2008).
Classically, the first phase or phase I is used to determine the safety, pharmacokinetics, pharmacodynamics and whether the predictions from prior animal and cell experiments apply to human use. Up to 100 usually healthy people (for highly toxic substances like cytostatics sick patients are examined) are treated with the first-in-human dose generated in preclinical studies. One possible approach to determine this dose is to use the No Observed Adverse Effect Level (NOAEL) from animal studies and factoring in a safety factor (usually 10) and a species conversion factor based on body surface. Applied dosages are increased step by step until significant adverse effects occur. Simple galenic formulations, e. g. solutions are usually used. At this point little data is available on the teratogenic potential of the API which is why male test subjects are often preferred (Becker et al., 2017).
Phase II is a randomized, usually placebo-controlled study incorporating up to 500 sick patients. The purpose of this stage is to gain data about the pharmaceuticals' therapeutic effect, adverse effects and toxicity. This is needed to perform dose-finding. As our proposed nose-to-brain delivery of liposomes is a novel approach, the scope of this stage is expected to be vast. Stage II is also known for having the highest failure rate among clinical stages (Becker et al., 2017).
Phase III studies are usually randomized, double-blinded, controlled and placebo-controlled studies incorporating several thousand patients. The larger testing group makes it possible to detect rarer adverse effects, drug interactions and effects in patient groups of different genetic equipment. Furthermore, the risk-benefit ratio is determined and the efficacy and innocuousness are refined (Becker et al., 2017). As HSV induced encephalitis is a rare disease, recruiting this many patients poses a logistical challenge.
Stage IV trials are not part of the original approval. Instead, they are performed after market launch for several years on an unlimited patient collective. Only these trials enable capturing of extremely rare adverse effects. It is also crucial for comparison with other therapies (Becker et al., 2017).
After successful clinical trials I to III, which take several years, and approval by authorities it is time to introduce our product to the public. As seen after the introduction of COVID-19 vaccination many people are skeptical about novel medications (Padda et al., 2022). As only four siRNA-based drugs are currently on the market, with the first approved in 2018, siRNA based drugs are still exotic and might evoke reluctance in certain population groups. However, due to the huge informational campaigns to advocate mRNA-based vaccines the public has been accustomed to nucleic acid drugs. This might pose an important factor for market acceptance.
Another crucial point is that the superiority of this product compared to the classic therapies would have to be proven for it to be reimbursed by health insurances. As stated above this product offers several advantages over standard acyclovir therapy. For Germany the Institute for Quality and Efficiency in Health Care (Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen, IQWiG) is responsible for this evaluation.