Exploring the Utility of ssDNA Aptamers Directed against Snake Venom Toxins as New Therapeutics for Snakebite Envenoming

Abstract

Snakebite is a neglected tropical disease that causes considerable death and disability in the tropical world. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapy for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions. In this study, we sought to explore the potential of ssDNA aptamers as toxin-specific inhibitory alternatives to antibodies. As a proof of principle model, we selected snake venom serine protease toxins, which are responsible for contributing to venom-induced coagulopathy following snakebite envenoming, as our target. Using SELEX technology, we selected ssDNA aptamers against recombinantly expressed versions of the fibrinogenolytic SVSPs ancrod from the venom of C. rhodostoma and batroxobin from B. atrox. From the resulting pool of specific ssDNA aptamers directed against each target, we identified candidates that exhibited low nanomolar binding affinities to their targets. Downstream aptamer-linked immobilised sorbent assay, fibrinogenolysis, and coagulation profiling experiments demonstrated that the candidate aptamers were able to recognise native and recombinant SVSP toxins and inhibit the toxin- and venom-induced prolongation of plasma clotting times and the consumption of fibrinogen, with inhibitory potencies highly comparable to commercial polyvalent antivenoms. Our findings demonstrate that rationally selected toxin-specific aptamers can exhibit broad in vitro cross-reactivity against toxin isoforms found in different snake venoms and are capable of inhibiting toxins in pathologically relevant in vitro and ex vivo models of venom activity. These data highlight the potential utility of ssDNA aptamers as novel toxin-inhibiting therapeutics of value for tackling snakebite envenoming.

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“…They are more advantageous compared to antibodies, such as low production cost, high thermal stability, and easy chemical modification. The single-strand oligonucleotide aptamers undergo unique conformation change in the presence of target analytes and bind with high affinity and specificity [ 23 , 24 ]. In vitro screening of aptamers by systematic evolution of ligands by exponential enrichment (SELEX) methods is commonly used against the target molecules.…”

“…In vitro screening of aptamers by systematic evolution of ligands by exponential enrichment (SELEX) methods is commonly used against the target molecules. Numerous aptamers have been selected against different kinds of targets, ranging from small metal ion to large whole cell with high affinity and specificity [ 24 , 25 , 26 , 27 ]. Aptamers are ideal candidates as a recognition element in biosensor development that bind the target with high affinity followed by translation and measurable signal via a transducer.…”

De novo designed proteins neutralize lethal snake venom toxins

“…Additional issues arise due to delayed administration of antivenom treatment 13 . Antibody 14,15,16,17,18,19,20,21,22 and non-antibody-based therapeutics 23,24,25,26,27,28,29,30 have been tested in preclinical studies, but the development of these types of molecules require either immunization of animals or development of large libraries that require extensive selection, screening, and optimization efforts 31 .…”

High throughput identification of human monoclonal antibodies and heavy-chain-only antibodies to treat snakebite

“…Various approaches are currently being used in the discovery and development of next-generation antivenoms, including drug discovery, aptamer selection, design of decoy receptors and monoclonal antibody discovery ( Chen and Murawsky, 2018 ; Laustsen, 2018 ; Laustsen et al, 2018b ; Clare et al, 2021 ; Albulescu et al, 2019 , 2020 ; Alomran et al, 2022 ). One of the most promising developments in antivenom research has been the identification of toxin-specific human monoclonal antibodies.…”