Genetically encoded biosensors for the non-invasive monitoring of the production of recombinant neurotoxins

Botulinum neurotoxins (BoNTs) are naturally produced by the anaerobic bacteria Clostridium botulinum and currently, there are seven different known serotypes of the toxin. BoNTs are known to cause botulism, a muscle paralysis illness in humans; botulinum neurotoxins are amongst of the most toxic substances known, as concentrations as low as 1ng/kg are proven to be fatal to humans (Sorouri et al., 2017). Amongst all, the BoNT serotype A is known to present the most danger to mankind, due to its extreme potency, combined with ease of production via simple biochemical synthesis. Hence, BoNT/A is classified as a type A bioterrorism agent by the Centre for Disease, Control and Prevention (Arnon et al., 2001). Despite its toxicity, BoNT/A has recently been exploited in medicine, for the treatment of disorders characterised by incorrect muscle contraction, such as dystonia, stroke, multiple sclerosis, hemifacial spasms, focal spasticity, hyperhidrosis (Laing, Laing & O'Sullivan, 2008) and aesthetic medicine, like the application of injections to reducing skin wrinkles (Liu et al., 2012).
BoNTs used in medical applications are based on recombinant toxin production; a key example is the biological drug DYSPORT, manufactured by Ipsen Pharma for the treatment of musculoskeletal or smooth muscle disorders in patients with neurological disease (Pharma, 2019). DYSPORT is based on the native activity of BoNT serotype A neurotoxins, which cause reduced transmission of nerve impulses to the muscle and in turn, this helps to relieve muscle hyperactivity. In-vivo, botulinum neurotoxins act as zinc metalloproteases which target and hydrolyse different proteins in the SNARE-complex. Cleavage of the SNARE proteins leads to blockage of neurotransmitter activity, which causes paralysis of the peripheral nerve terminals (botulism); in turn, this leads to breathing difficulties or death (Sikorra et al., 2016).
There are several challenges arising from bioprocessing of recombinantly produced toxins, such as BoNTs. Due to the high toxicity, production needs to be done under biosafety conditions, which limits the quality and quantity control measurements that can be done to test the final product (Pharma, 2019). Even when such measurements are achieved, off-line sampling methods are used, which means that testing is slower and low throughput. Currently, scientists are attempting to optimise production processes of recombinant neurotoxins, in order to improve yield and quality. Other studies are also trying to improve the pharmaceutical applications of BoNTs by extending the efficacy of neurotoxin-based drugs and improving their localisation, in order to reduce off-target activity (Rossetto, Pirazzini & Montecucco, 2015). Such studies require bioprocess optimisation which involves testing how different parameters affect all bioprocessing steps. Therefore, there is a demand for in-vivo, high throughput testing of samples to support a faster development and optimisation of the bioprocessing and manufacture. This can be achieved through a series of genetically encoded biosensors which will speed up the detection process, as biosensors allow for a simpler, non-invasive measurements of key parameters.
Genetic biosensors can be used as in-vivo analytical devices that detect a biological response and couple it to a fluorescence output. Various types of biosensors exist, which can be used to monitor of the current challenges arising from recombinant BoNTs expression, in a robust and cost-effective way. (Eivazzadeh-Keihan et al., 2018). As a long-term plan, other biosensors can be used to monitor production of recombinant BoNTs, such as transcription-based biosensors as an indication of productivity rates, or FRET-based biosensor, used to monitor neurotoxin activity through tracking the binding and cleavage of SNAP-25 domains (Rossetto, Pirazzini & Montecucco, 2015); one of the native targets of BoNT/A.