Polymamba

Polymamba: DNA Origami-Aptamer Complexes as Novel Treatment for Snakebites Overview Although seldom discussed in literature, the global burden of snakebite envenomation plagues the vast majority of tropical areas (Kasturiratne et al., 2008). The lack of accurate and specific epidemiological quantification of this condition, coupled with stagnant progress in the supply, accessibility, and effectiveness of current antidotes renders it a matter of great urgency on the global health agenda. The current standard of care consists of intravenous (IV) administration of animal-derived antivenoms, which often lack adequate safety profiles and universal effectiveness in toxin neutralization (Gutiérrez et al., 2006). Even more problematic is the worldwide supply shortage of these treatments: due to the termination of antivenom production by large pharmaceutical companies such as Sanofi Pasteur, experts predict that current stockpiles will be exhausted by June of 2016 (Schiermeier, 2015). Thus, the critical nature of this global health issue demands a call to action for a cheaper, more effective venom neutralizer that can evade the high costs and inefficient production processes of current treatments.

Epidemiology According to a 1998 study conducted by the World Health Organization (WHO), the annual incidence of snakebite exceeds 5 million people on a global scale (Chippaux, 1998).

Coupled with information extracted from databases maintained by the United Nations (UN), World Bank (WB), and Food and Agriculture Organization (FAO), these numbers are estimated to be between 1.2 million and 5.5 million annually (Kasturiratne et al., 2008). The regions of highest affect include South and Southeast Asia and Sub-Saharan Africa (which will be designated the “target regions”). Generally, incidence of snakebite is correlated with the prevalence of a venomous species in a particular region – the Malayan pit viper A. rhodostoma in Southeast Asia,

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for instance, or the saw-scaled viper E. carinatus in Africa (Reid & Theakston, 1983). Given an envenomation rate of approximately one in every four snakebites, the annual envenomings in these regions is approximately 300,000 in its low range estimate (Kasturiratne et al., 2008). Due to a lack of adequate healthcare management and access to treatment in these regions, the mortality rate is proportionally higher than in other areas, resulting in approximately 20,000 deaths per year (Kasturiratne et al., 2008). Aside from fatality, victims reportedly suffer from a number of other debilitating morbidities, including amputation, chronic ulceration, paralysis, and even blindness (Brown, 2012).

Pathophysiology Once the snake venom enters the blood of its victim, its path of action is rapid and potent.

The effects of poisoning are predominantly hemostatic and are initially localized to the area of the bite, causing swelling within minutes to hours of envenomation depending on the species (Reid & Theakston, 1983). As time elapses, systemic absorption of the venom may take place, which can be elucidated through symptoms such as vomiting, abdominal pain, and collapse of the victim.

Once the venom diffuses from local to systemic circuits, its region of affect moves beyond the hemostatic system and manifests itself through neurototic, myotoxic, cardiac, and renal effects.

Hemorrhagic toxins are most commonly detected and can cause spontaneous oozing (due to endothelial damage), intravascular coagulation, or hypovolemic shock due to the rapid diffusion of blood into the swollen limb; these effects are often lethal and can lead to rapid degeneration if not immediate death. Neurotoxins generally inhibit cholinergic function, impairing proper neuromuscular activity, and myotoxins exert a generalized effect across the skeletomuscular system. Systemic signs of envenomation can take place as early as 20 minutes in some snakes but

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most often don’t appear until several hours after the initial bite. During this time, however, the venom can circulate through and cause damage in multiple systemic routes.

Current Standard of Care Due to the definitive nature of the incident, all victims can ascertain whether or not they have been bitten and generally seek basic first-aid treatment regardless of confirmation of envenomation. This includes cleaning the site of incision and applying bandages or other forms of compressive gauze over the wound (Reid & Theakston, 1983). Depending on the availability of access to health clinics, victims are then treated with antivenom intravenously after testing for systemic symptoms. Confirmation of envenomation is necessary due to th highly immunogenic profile of antivenom. In most developing countries (i.e. the regions of highest affect), the standard form of therapy is a polyvalent antivenom treatment that dismisses the need for rapid diagnostic tests to identify the snake species (Brown, 2012). 100 mL of antivenom (half-life = 80 – 100 hours) is administered as an initial dose, and patients are monitored every 6 hours for their systemic bleeding and blood coagulation levels (Mohapatra & Mohanty, 2010). If necessary, additional doses of 50 – 100 mL doses are given until complete correction occurs (Vijeth et al., 2000). If effective, antivenom injections can reverse the effects of systemic poisoning even days after the bite, but inefficient production processes and substandard quality in target regions lead to a highly ineffective treatment process (Reid & Theakston, 1983).

Antivenom Evaluation Most current polyvalent antivenoms are produced by extracting snake venom for immunization in horses or sheep, from which the antibodies are collected in the form of serum (Morais & Massaldi, 2006). This protocol of immunotherapy has been more or less in place since the 1930s; no major improvements or discoveries have been made since then (Brown, 2012).

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Although simple in concept, this process greatly depletes both temporal and monetary resources.

It requires the maintenance of serpentariums for venom extraction as well as farmsteads of horses and sheep for immunization, which inhibits accessibility to large economies of scale. Furthermore, the immunized plasma must undergo transport to processing plants for purification and extraction of the final product, which must then be maintained in specific storage conditions (2 – 8 degrees Celsius) until use (Wolf et al., 2011).

Due to the steep costs and time needed for antivenom production, many companies are facing low profitability and subsequently terminating their antidote production in pursuit of other projects. Sanofi Pasteur, a pharmaceutical company based in Lyon, made the internal decision in 2010 to cease production of Fav-Afrique, a pan-African polyvalent antivenom that neutralizes 10 different African species. Fav-Afrique is currently unrivaled in its potency and polyspecificity, and sources from Doctors Without Borders say that the last batch will be drained by mid-2016 (Schiermeier, 2015). Sanofi’s decision to exit the market was driven by the cheaper pricing of competitor pharmaceutical companies based in countries such as South Africa, India, Mexico, and Costa Rica. However, these ‘knockoffs’ have yet to establish their effectiveness in patient treatment, and none have proven the level of polyvalency of Fav-Afrique. In fact, the quality of antivenom used in sub-Saharan Africa varies widely across manufacturers, and recently published reports indicate that the two dominant products in the African market (accounting for up to 90% of treatment use) is highly ineffective against the species they claim to target (Brown, 2012). Thus, the combination of antivenom shortage and the decline of quality products results in a dismal effective treatment rate of 2.5% in some target regions.

Moreover, even in the case of effective therapy, all antivenoms face the drawback of potential adverse reactions after administration. According to the WHO, approximately 20% of

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victims injected with antivenom will experience allergic reactions, usually as an immunogenic response to horse serum (Warrell, 2005). These reactions can be characterized in the form of early anaphylactic reactions, the direct activation of the complement system by the serum antibody, or pyrogenic reactions, the pyrogenic contamination of the antivenom-serum complex (Mohapatra & Mohanty, 2010). Although the adverse effects are typically mild, including nausea, diarrhea, or tachycardia, a minority of patients do experience severe bouts of hypertension and can even develop fatal anaphylaxis. These effects are compounded in polyvalent therapies due to the multiplication of distinct antibody identities in the immunized serum. Even if the market for quality antivenom products were to resurface, that does not eliminate the need for a product with a reduced profile of side effects.

Polymamba: A Novel Technology Platform The Polymamba technology platform strives to address this dearth of potent, cost-effective snakebite treatments by engineering a DNA-origami poly-aptamer complex with the ability to specifically target and neutralize a wide range of venomous toxins. The complex consists of a DNA origami nanostructure that serves as the delivery vehicle; attached to the DNA origami scaffold are single-stranded oligonucleotides called aptamers that confer highly specific molecular recognition of toxin targets (Jayasena, 1999). To engineer this complex, a long, viral “scaffold” strand is hybridized to shorter “staple” strands of oligonucleotides to create handles that bind the aptamers themselves (Marchi et al., 2014). These complexes are then suspended in sterile saline solution for IV injection into the site of the bite to ensure efficient systemic delivery (Chippaux, 1998). Intramuscular (IM) delivery is also possible if intravenous administration is not feasible.

Once in the body, these aptamers will neutralize the toxins released by envenomation through recognition and binding mechanisms.

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The use of DNA as a scaffold for aptamers allows for a highly specified route of distribution, evading the consequences of systemic toxicity that are present in many other nanoparticle delivery models (Zhang et al., 2014). Furthermore, DNA nanostructures exhibit high biocompatibility and are surprisingly stable, even under harsh thermal and chemical environments (Kim et al., 2014). This creates a larger application window for DNA origami, which is significant given the extreme physiological changes that take place in the body after envenomation.

Furthermore, the programmability of scaffold generation through precise intermolecular interactions results in a sharp increase in the available surface area that can be utilized for multifunctional activity (Marchi et al., 2014). The customizability of DNA as a delivery vessel lends flexibility to its engineering design, allowing it to hold a multitude of different aptamer structures that can target a wide range of toxins.

Likewise, the decision to use aptamers rather than antibodies in target recognition is largely due to its stability and specificity. Like antibody proteins, aptamers can undergo denaturation under harsh conditions, but this process is rapidly reversible. Moreover, aptamers are much more stable at room temperature and don’t require supplementary cooling during transportation, rendering it a much higher ex vivo shelf life (Jayasena, 1999). In terms of structure, aptamers can take on a much higher permutation of shapes and structural variants, which is largely due to the interaction forces between nucleotide bases. As a result, aptamers can be engineered to a much wider database of target molecules. Furthermore, the binding affinity and specificity of aptamers rival and even sometimes surpass that of antibodies. The dissociation constants range from picomolar to the low nanomolar range, resulting in a very slow separation time in the bound aptamer-protein complex (Talbot et al., 2011). Furthermore, aptamers have an elimination halflife of approximately 8 hours (only when given IV), which is comparable to the pharmacokinetic

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profile of antivenom. Finally, because they don’t depend on bacterial or animal cell culture, aptamers are much easier to produce and engineer to economies of scale (Stoltenburg et al., 2007).

Due to its use of different animals or bacterial strains between each production process, antibody production is easily subject to batch-to-batch variation. Aptamers, on the hand, are generated through chemical synthesis, which creates highly accurate and reproducible constructs that are further purified under denaturation (Jayasena, 1998).

Immunomodulation Aside from neutralizing target toxins, the DNA origami scaffold can also activate immunostimulation through the coating of cytosine-phosphate-guanine (CpG) structures. Like aptamers, CpG sequences can be hybridized onto the DNA origami scaffold by binding to singlestranded handles that complement the anchor strand of the CpG sequence. In humans, unmethylated CpG sites stimulate toll-like receptor 9 (TLR9) activation on B lymphocytes, inducing a humoral immune response in the body (Bekeredjian-Ding & Jego, 2009). Schüller et al. demonstrated the potential of DNA origami constructs as an effective carrier of CpG oligonucleotides to induce immunostimulation (2011). Results showed that the eventual uptake of CpG strands by macrophages was much higher and more rapid when they were conjugated to a DNA origami scaffold as compared to other DNA nanostructures. This effect demonstrates the importance of size and compactness of the scaffold in contributing to the immunomodulatory pathway.

Specifically, the process of toxin clearance begins when endosomal TLR9 proteins recognize the unmethylated CpG sequences on the DNA origami-aptamer complex. TLR9 is part of a family of TLR receptors that are involved in the recognition of microbial antigens such as CpG motifs, which are prevalent in bacterial genomes but are rarely present in vertebral genomic

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DNA (Krieg, 2002). The activation of TLR9 on the surface of a B lymphocyte results in the upregulation of activation marker C-type lectin CCD69, resulting in cytokine release and the secretion of antibodies (Bekeredjian-Ding & Jego, 2009). Antibody binding to the CpG motifs then triggers the complement cascade by recruiting effector cells to interact with the FC region.

This last step induces phagocytosis and neutralization of the antigen – in this case, the venomous toxin (Doyle et al., 2004).

Apart from the adaptive immune response triggered by B lymphocytes, TLRs have also been shown to be directly linked to the macrophage-mediated phagocytosis of antigens (Doyle et al., 2004). This alternative immune response involves the activation of TLRs that appear on the surface of the macrophage cells themselves. TLR activation initiates a signal transduction cascade that results in the upregulation of scavenger receptors, which leads to a significant increase in phagocytic activity. These results lead researchers to believe that the innate immune response possesses a level of specificity that is due in part to the specificity of TLR recognition of foreign antigens; in this case, the recognition of CpG sequences on the DNA origami-aptamer complex by TLR9 (Akira, 2011).

Technology Production The generation of Polymamba relies on the protocols of SELEX and CaDNAno for the engineering of DNA origami and aptamer strands, respectively. SELEX stands for the Systematic Evolution of Ligands by EXponential Enrichment and consists of a comprehensive screening of random combinatorial oligonucleotide sequences to isolate and amplify the sequences with the highest binding affinity for any given target (Song et al., 2012). The screening process begins with the incubation of a library of approximately 1012 – 1015 randomly selected oligonucleotide sequences, during which only a small fraction of these sequences bind to the given target. The

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unbound sequences are separated from the bound sequences in an elution step through nitrocellulose filter portioning, and the selected sequences are then amplified through PCR to generate a new library (Jayasena, 1999). This process is repeated until a desired affinity is reached, generating an evolutionarily optimal sequence that can actively and specifically bind to the target molecule.

In order to confer polyvalency, venom will be extracted from the snakes deemed of medical importance in the target regions. In Africa, these snakes include back-fanged snakes Colubridae, African spittin cobras Naja, and puff adders Bitis arietans (Warrell, 2010). Among the medically significant, venoms containing abnormally high amounts of endonucleases, such as Bothrops and C. adamanteus, will be excluded from the selection process due to their high cleavage activity of polynucleotides (Richards et al., 1965). The selected venoms will then be purified for their library of toxins, which largely fall into the classes of neurotoxins, myotoxins and hemotoxins (Reid & Theakston, 1983). The process of SELEX is run on the finalized list of toxins, creating a pool of aptamers that will bind to these toxic targets in the human body. Lauridsen et al. demonstrated the viability of a one-step selection method for aptamer generation against alpha-Bungarotoxin, a toxic component in the venom of krait snake (Bungarus multicinctus), confirming the feasibility and efficacy of aptamer generation as a therapeutic against envenomation (2011).

Like SELEX, the CaDNAno protocol dictating DNA origami assembly is fairly straightforward and is, in this case, software-mediated (Castro et al., 2011). The ideal design for a DNA origami structure consists of a 6-kilobase base “scaffold” strand and hundreds of short “staple” strands, holding the highly complex scaffold in place (Rothemund, 2006). Each toxin will necessitate a multitude of aptamers specific to that molecule; thus, the number of scaffold strands needed will depend on the number of toxins in the target pool. Due to the complexity of the

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Polymamba complex, rectangular packing is preferable to honeycomb lattice packing in order to allow for more densely packed aptamers (Castro et al., 2011). Before initiating the DNA origami building process, the feasibility and shape of the CaDNAno design can be visualized through the Computer-aided engineering for DNA origami (CanDo) tool. Both scaffold and staple sequences can be purchased through commercial vendors, although the latter may require some purification steps to remove truncated synthesis pieces. After oligonucleotide preparation is complete, they are pooled together and run in molecular self-assembly reactions to create the actual DNA origami construct.

Polymamba Evaluation Compared to current standards of antivenom treatment, Polymamba poses many clear advantages. It exhibits a low immunogenicity profile due to the in vitro chemical synthesis of oligonucleotides (Schüller et al., 2011). This not only minimizes the risk of adverse reactions but also allows for immediate injection upon incidence of the bite without waiting for confirmation of envenomation. Due to the rapid kinetic profile of venom within the human body, generating adverse localized and systemic effects within minutes, this expeditious response can serve to neutralize the toxins before further harm is done (Reid & Theakston, 1983). In addition, oligonucleotide constructs are highly stable in both ambient and harsh environments, which allow them to stay in the cytoplasm long enough to carry out their defined functions. They also show virtually no detectable toxicity while maintaining a high level of specificity for the target toxin molecules (Schüller et al., 2011).

Aside from its pharmacokinetic advantages, Polymamba also exhibits clear production efficiencies and commercial advantages. Because the technology relies wholly on in vitro chemical synthesis and sidesteps the need for in vivo animal immunization, the product is rapidly scalable

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and can be generated within a much more condensed timeframe. In addition, the costs for production are much lower because they only involve the selection and generation of certain oligonucleotide constructs, avoiding the bulky costs of animal maintenance and immunization, antibody purification, and product transportation (Morais & Massaldi, 2006). Finally, the potential pan-African and pan-Asia polyvalency of Polymamba allows it to be effectively used in all clinical settings, dismissing the need to develop specific and distinct immunotherapies for each venomous species.

Competitive Landscape Aside from current antivenom manufacturers, the market landscape includes a potential competitor, Operational Technologies Corporation (OpTech), that is also focusing on aptamer technology in its development of treatment for reptile venoms. OpTech proposes to test the efficacy of aptamers against various venoms and attach these aptamers to dendritic scaffolds to slow aptamer degradation in the body (Bruno, 2015). Like DNA origami, DNA dendrimers as nanoparticle carriers can serve as highly programmable scaffolds that exhibit biostability, high specificity and affinity, and high structural variability (Zhang et al., 2015). The use of DNA scaffolding is an undeniable upgrade from the creation of an “aptamer soup” that is highly susceptible to in vivo degradation and inefficient mobilization (Pednekar et al., 2012). However, dendrimers pose a host of drawbacks compared to DNA origami: notably, they are much smaller in size, which can lead to rapid renal excretion, limiting its therapeutic potential in the bloodstream (Fadeel et al., 2015). In addition, dendritic scaffold generation is highly costly, and most studies of dendrimers have only been shown to be conducted on a relatively small scale. From a manufacturing standpoint, then, these aptamer-dendrimer constructs don’t exhibit a distinct costeffective advantage over antivenoms. Furthermore, they lack a specific immunostimulant factor

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that induces activated phagocytosis by the adaptive immune system; rather, they seem to rely on the natural clearance of the antigen by the innate immune routes of the body. As of 2014, OpTech has submitted a grant proposal to initiate Phase I trials for its aptamer-dendrimer constructs, indicating that it is still in the nascent stages of clinical approval. Due to the product’s comparatively weak pharmacokinetic profile and commercial production strategy, there lacks a compelling reason to believe that OpTech’s product will be a major competitive threat to Polymamba (Bruno, 2015).

Intellectual Property Strategy The two major technologies to include in Polymamba’s intellectual property (IP) strategy is the use of SELEX and DNA origami devices. The former is filed under a Gold et al. 2002 patent that specifies the method for generating nucleic acid ligands through evolutionary selection. In 2001, it was exclusively out-licensed by Gilead Sciences to Archemix Corporation; the latter retains all therapeutic and commercial rights to the SELEX process (Gilead, 2001). Since then, Archemix has undertaken a number of sublicensing agreements with biotech companies that extend SELEX applications to therapeutic fields outside of its core business. For instance, in 2002, a worldwide exclusive sublicense was granted to Germany-based NOXXON Pharma for the use of SELEX in combination with NOXXON’s technology for developing oligonucleotide therapeutics (Temin & Gilbert, 2002). Polymamba will aim to negotiate a sublicensing agreement with Archemix that includes a partially exclusive license in the field of envenomation therapies for the purposes of potentially extending the technology to target other animal venoms in the future (NIH, 2015). Based on precedent transactions, licensing SELEX can exist in the form of an upfront cost ($1 – 5 million) or a profit-sharing contract with Archemix that grants an annualized royalty rate (Temin & Gilbert, 2002; Merck, 2007).

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Regarding the use of DNA origami, Bachelet et al. filed a 2002 patent application through Harvard College that defines the scope of use of DNA origami devices as biologically active carriers in the body. Although the patent has not yet been granted, approval is likely and will necessitate a licensing agreement (ideally one that is partially exclusive) with Harvard in the form of upfront payment, royalty payments, or equity shares. As with SELEX, Polymamba will aim to negotiate an agreement that allows for exclusive reign in the field of envenomation therapies to create a barrier to entry for potential competitors.

Clinical Strategy Polymamba’s preclinical strategy involves conducting in vivo preclinical trials on rats, in accordance to the WHO guidelines for antivenom testing (WHO, 2010). In order to test the efficacy of the aptamer complex in neutralizing the overall toxic profile of the venom, each aptamer will need to undergo the venom-neutralizing potency test (WHO, 2010). After individual quality assessment, the entire Polymamba construct should be tested for its preclinical neutralizing potency to assure routine quality control. Furthermore, the median lethal dose assay and the median effective dose assay should be conducted to determine the appropriate amount and frequency of dosing (Gutiérrez et al., 2013). Due to the polyvalent and condensed nature of the antidote, dosage is predicted to be much lower than is needed for antivenom treatments (Bruno, 2015). Preclinical endpoints should also include establishing the stability of the Polymamba construct both in vivo (rate of degradation) and ex vivo (shelf life). If the concentration of endonuclease molecules is particularly strong in the designated venoms, aptamers can be modified to include analogs that increase their nuclease resistance; these modified constructs can then be tested against the venoms with high DNAse activity using the tests detailed above (Lauridsen et al., 2012).

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Clinical trials, on the other hand, will focus on safety and detecting immunogenicity as well as measuring the efficacy of Polymamba in human patient pools as compared to the standard of care (antivenom). Trials will be conducted within the target regions in health centers that meet the following requirements: 1.) trained health personnel and regulated management, 2.) high incidence of snakebite in the area that will ensure sufficient recruitment, and 3.) dense infrastructure of clinics that can allow for comparability of results (Chippaux et al., 2007). Major efficacy endpoints include blood coagulability and improvement of neurotoxicity due to their association with the symptomatic effects of envenomation (Müller et al., 2012). Safety measurements will include detecting unanticipated adverse events as well as evaluating various levels of dosing, as humans may react differently to dosage than rats.

Costs and Pricing Polymamba will be priced at a multiple to the production cost and will be benchmarked against current competitors in the target regions, which currently range from $55 to $640 per effective treatment (Brown, 2012). Production costs of Polymamba include purchase of oligonucleotide libraries as reagents as well as packaging costs that include saline suspension solution and antidote storage containers (Schmidt et al., 2015). Engineering costs are negligible after licensing contracts have been solidified, and the use of CaDNAno to design DNA constructs is free of charge (Castro et al., 2011). The total per unit cost based on these cost factors can be estimated to be about ~$10 per effective treatment, which may even be an overestimate due to the low dosage that is required of the dense polyvalent aptamer construct. Thus, even if Polymamba prices at a 40% discount to its lowest priced competitor, the per treatment sales of $35 will still be 3.5x the initial production cost, generating a high profit margin. Based on the relatively unsaturated

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market landscape and the demand for a higher quality treatment, Polymamba sales can be expected to peak at $4 – 5 million annually.

Commercial Strategy Partnership with a research incubation program will ensure low upfront costs and managerial and financial support throughout the stages of R&D and commercialization. Many of these incubators require an equity ownership in exchange for their resources, but given the frontloaded timeline of Polymamba’s costs (e.g. licensing, R&D), this is the best option for the company (Kirkpatrick, 2015). Initial funding can also be secured through government-funded agencies such as the NIH, private foundations such as the Gates Foundation, and biotech angel investors. Given the urgency of the snakebite epidemic, it is also feasible to establish a publicprivate partnership by recruiting various national and regional health organizations and private foundations to provide the necessary financial resources and distribution networks (Gutiérrez et al., 2006). Finally, partnership with the various Ministry of Health departments in the target regions will ensure market penetration and a widespread distribution infrastructure. It is the goal of Polymamba to directly target and reverse the pandemic snakebite crisis affecting so many people in the world today.

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