A new broad acting antidote for venom-induced injury including local tissue damage and / or skin irritation

Abstract

Provided herein are methods of treating or preventing venom induced injury, including local tissue damage and / or skin irritation using composition comprising an effective amount of heparin and / or heparinoid. Also provided are uses of said composition in the manufacture of a medicament for treating or preventing the same and said composition for use in treating or preventing the same.

Description

A NEW BROAD ACTING ANTIDOTE FOR VENOM-INDUCED INJURY INCLUDING LOCAL TISSUE DAMAGE AND / OR SKIN IRRITATIONField

[0001] The present invention relates to methods for treating or preventing venom induced injury including tissue damage and / or skin irritation.Reference to a Sequence Listing

[0002] The present application contains a sequence listing which has been submitted electronically as an XML document in the ST.26 format and is hereby incorporated by reference in its entirety. Said XML copy, created on 5 March 2024, is named “P0063826AU Seq Listing. xml” and is 22.9 KB in size.Background

[0003] Interaction with venom from various animals and plants can induce injury, local tissue damage and / or skin irritation. For example, the bluebottle, a species of siphonophore that resembles a jellyfish are native to the surrounding waters and are the cause of the most “jellyfish” stings in Australia. An estimated 10,000 envenomation events caused by bluebottle “jellyfish” occurred each summer. Globally, the closely related Portuguese man o' war are a major concern. Moreover, species of true jellyfish, such as sea nettles, are widely distributed across the Atlantic, Pacific and Indian Oceans and its stings cause a mild to moderate pain, with severe cases requiring medical attention.

[0004] Further to the above, snakebites kill an estimated -138,000 people each year, with another -400,000 people experiencing devastating long-term morbidity. Most of these envenomings occur in Sub-Saharan Africa and South / Southeast Asia, with young adults and children disproportionately impacted. This makes snakebite envenoming the deadliest of the neglected tropical diseases (NTDs) with its burden landing mainly on impoverished rural communities. The resulting annual disease burden from snakebite in West Africa and Southeast Asia alone amounts to -319,000 and -392,000 disability adjusted life years (DALYs), respectively, with associated costs for the latter (2.5 billion USD) representing -0.1% of the 15 region’s GDP. Consequently, the World Health Organization (WHO) recently elevated snakebiteto a ‘priority category A NTD’ and announced the ambitious goal of reducing the global burden of snakebite in half by 2030.

[0005] Current antibody based antivenom treatments are species-specific, rely on a cold-chain, and require intravenous administration in hospital settings. Moreover, antivenoms can induce adverse reactions, and are often prohibitively expensive even if available. Crucially, antivenoms are ineffective against severe local envenoming, which involves painful progressive swelling, blistering and / or tissue necrosis around the bite site and can lead to loss of limb function, amputation, and lifelong disability. Accordingly, there is a need to develop an alternative new broad acting antidote or composition for treating or preventing venom induced injury, including local tissue damage and / or skin irritation.

[0006] To meet this target, a basic molecular understanding of how diverse venoms interact with human physiology is required to inform the development of new therapeutics. Here the inventors use a functional genomics approach to define venom / target genetic interactions that modify cytotoxicity and use this information to develop a novel and local acting venom antidote.

[0007] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0008] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.Summary of Invention

[0009] Using a functional genomics approach to define human genes that genetically interact with venoms from various species of animals and plants (such as bluebottle and sea nettle jellyfish venoms and spitting cobra venoms), the inventors of the present application have surprisingly found that most genes that confer resistance to venom cytotoxicity control proteoglycan biosynthesis, suggesting heparin and heparinoids as possible inhibitors. In the case of cobra venom, the inventors also surprisingly found that heparinoids prevent venom cytotoxicity by inhibiting three finger cytotoxins.

[0010] According to a first aspect, the present invention provides a method of treating or preventing venom induced injury including local tissue damage and / or skin irritation which comprises administering to a subject a composition comprising an effective amount of heparin and / or heparinoid.

[0011] According to a second aspect, the present invention provides use of a composition comprising an effective amount of heparin and / or heparinoid in the manufacture of a medicament for treating or preventing venom induced injury including local tissue damage and / or skin irritation.

[0012] According to a third aspect, the present invention provides a composition comprising an effective amount of heparin and / or heparinoid for use in treating or preventing venom induced injury including local tissue damage and / or skin irritation.

[0013] Numbered statements of invention are as follows:1. A method of treating or preventing venom induced injury including local tissue damage and / or skin irritation which comprises administering to a subject a composition comprising an effective amount of heparin and / or heparinoid.2. Use of a composition comprising an effective amount of heparin and / or heparinoid in the manufacture of a medicament for treating or preventing venom induced injury including local tissue damage and / or skin irritation.3. A composition comprising an effective amount of heparin and / or heparinoid for use in treating or preventing venom induced injury including local tissue damage and / or skin irritation.4. The method according to statement 1, the use according to statement 2, or the composition according to statement 3, wherein the venom comprises three-finger toxins (3FTxs) and / or one or more other toxin.5. The method, the use, or the composition according to statement 4, wherein the three- finger toxins (3FTxs) are three finger toxin cytotoxins (or cardiotoxins).6. The method, the use, or the composition according to statement 4, wherein the other toxins are toxins that interact with or stick to heparin and / or heparinoids.7. The method according to any one of statements 1 or 4 to 6, the use according to any one of statements 2 or 4 to 6, or the composition according to any one of statements 3 to 6, wherein the heparinoid is a low molecular weight (LMW) heparinoid.8. The method, the use, or the composition according to statement 7, wherein the LMW heparinoid is a tinzaparin, dalteparin, enoxaparin, or a pharmaceutically acceptable salt thereof.9. The method according to any one of statements 1 or 4 to 8, the use according to any one of statements 2 or 4 to 8, or the composition according to any one of statements 3 to 8, wherein the composition is administered to a skin of the subject.10. The method according to any one of statements 1 or 4 to 9, the use according to any one of statements 2 or 4 to 9, or the composition according to any one of statements 3 to 9, wherein the composition is administered topically, subcutaneously, or transcutaneously.11. The method according to any one of statements 1 or 4 to 10, the use according to any one of statements 2 or 4 to 10, or the composition according to any one of statements 3 to 10, wherein the composition is administered in less than 5 minutes, less than 10 minutes, less than 30 minutes, less than 60 minutes, or about 60 minutes after exposure to the venom.12. The method according to any one of statements 1 or 4 to 11, the use according to any one of statements 2 or 4 to 11, or the composition according to any one of statements 3 to 11, wherein the composition is a topical cream formulation, a spray formulation, an injectable formulation, a sunscreen formulation.13. The method according to any one of statements 1 or 4 to 12, the use according to any one of statements 2 or 4 to 12, or the composition according to any one of statements 3 to 12, wherein the venom induced local tissue damage and / or skin irritation is dermonecrosis.14. The method according to any one of statements 1 or 4 to 13, the use according to any one of statements 2 or 4 to 13, or the composition according to any one of statements 3 to 13, wherein the venom originates from a siphonophore, jellyfish, an insect, a plant, or a snake.15. The method, the use, or the composition according to claim 14, wherein the siponophore is Physalia utriculus (blue bottle) or Physalia spp.16. The method, the use, or the composition according to statement 14, wherein the jellyfish is Chrysaora quinquecirrha (sea nettle).17. The method, the use, or the composition according to statement 14, wherein the insect is Pristhesancus plagipennis (assassin bug).18. The method, the use, or the composition according to statement 14, wherein the plant is Urtica ferox (New Zealand stinging nettle).19. The method, the use, or the composition according to statement 14, wherein the snake is a cobra selected from the group consisting of Nigerian Naja nigricollis (black-necked spitting cobra), Tanzanian Naja nigricollis (black-necked spitting cobra), Naja pallida (red-necked spitting cobra), Naja kaouthia (monocled cobra), Naja atra (Chinese cobra), and Naja naja (Indian spectacled cobra).Brief Description of Drawings

[0014] Preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0015] Figure 1 illustrates a whole genome CRISPR-Cas9 knockout screen for identifying genes required for African spitting cobra venom cytotoxicity. Figure 1A is a photo of Red spitting cobra (Naja pallida) and black necked spitting cobra (Naja nigricollis) and a map which illustrate their distributions. Figure IB is a graph illustrating HAP1 cell viability as determined by resazurin assays after 24 h treatment with serial dilutions of spitting cobra venoms (n = 3). Figure 1C is a schematic of pooled CRISPR knockout library screens. HAP1 cells were transduced with a whole genome knockout library at MOI = 0.3. Venom was added to library cells and genomic DNA extracted from selected and unselected control populations before undergoing next generation sequencing. Analysis was calculated using the MAGeCK pipeline. Figures ID and IE are graphs illustrating gene enrichment analysis of screens performed using MAGeCK. Horizontal dotted line indicates -loglO(false discovery rate) (FDR) = 1 and vertical dotted lines indicate log2(fold changes) (LFCs) of -2 and 2. Plots generated usingEnhanced Volcano (vl.10.0) R package. Figures IF and 1G are graphs illustrating top canonical pathways identified through Ingenuity Pathway Analysis (IP A).

[0016] Figure 2 illustrates that heparan sulfate biosynthesis is required for spitting cobra venom cytotoxicity. Figure 2A is a schematic representation of the heparan sulfate biosynthesis pathway. Figures 2B to 2D are graphs illustrating pools of single sgRNA knockout cells for heparan sulfate biosynthesis hits (XYLT2, B4GALT7, B3GAT3, EXTL3, EXT1, EXT2, NDST1, and SLC35B2') and a non-targeting control sgRNA (NTC) were generated via lentiviral transduction in HAP1 cells. Pooled knockout cells were treated with 10 pg / mL N pallida (Figure 2B), Nigerian (NGA) N. nigricollis (Figure 2C), or Tanzanian (TZN) N. nigricollis (Figure 2D) venom for 24 h and viability ascertained through resazurin. Significance determined by one-tailed Mann-Whitney test, *P<0.05, **P<0.01 (n = 4-5).

[0017] Figure 3 illustrates that heparin and its LMW variants block Naja venom action in vitro. Figure 3A depicts chemical structures of low molecular weight heparinoids, dalteparin and tinzaparin. Figure 3B is representative brightfield microscopy of HAP 1 cells after 24 h exposure to 10 pg / mL N. pallida or Nigerian (NGA) N. nigricollis venom, simultaneously treated with 20 pM heparin, tinzaparin or dalteparin. Figures 3C to 3E are graphs illustrating effects of simultaneous exposure of venoms and heparin or heparinoids on HAP1 cells. Venoms (10 pg / mL) and serial dilutions of heparin (H), tinzaparin (T) and dalteparin (D) (1.25-20 pM) were added simultaneously to HAP1 cells. Resazurin cell viability assays were performed after 24 h of treatment. Significance determined by 2-way ANOVA and Dunnett test, ****P<0.0001 (n = 3). Figures 3F is a graph illustrating effects of asynchronous exposure of NGA venom and heparin on HAP1 cells. HAP1 cells were treated with 10 pg / mL N. nigricollis (NGA) venom before addition of 20 pM heparin immediately after, or 5-, 10-, 30-, 60- or 90-min post venom application. Significance determined by Ordinary one-way ANOVA and Dunnett test, **P<0.01, ***P<0.001, ****P<0.0001 (n = 3).

[0018] Figure 4 illustrates that heparin binds 3FTxs and prevents their cytotoxicity. Figures 4A to 4C are representative flow cytometry histograms of WT HAP1 cells in gray and cells exposed to Alexa488-tagged N. pallida venom only (Figure 4A), venom with heparin (Figure 4B), or venom with tinzaparin (Figure 4C). Figure 4D is a graph depicting quantification of binding intensity (n = 5). Significance was determined by One-way ANOVA and Dunnett test, **P<0.01. Figure 4E is a graph depicting heparin affinity chromatography of N. pallida venom.Unbound (U), Peak 1 (Pl), Peak 2 (P2) and Peak 3 (P3). Figure 4F is a graph depicting cation exchange chromatography of Peak 3. Figure 4G is a photo of SDS-PAGE gel of whole venom and resulting toxin fractions. Figure 4H is Surface plasmon resonance (SPR) results. Representative normalized sensorgrams of toxin binding to tinzaparin. Figure 41 is fits of the SPR data from Figure 4H to a 1 : 1 binding model are shown and KDS are indicated on each plot. Figure 4 J is graphs depicting cytotoxicity of 10 pg / mL of each toxin fractions and rescue by 20 pM tinzaparin. Significance determined by 2-way ANOVA and Sydak test, ****P<0.0001 (n = 3). Figures 4K to 4M are graphs depicting cytotoxicity of venoms containing 3FTxs (Figure 4K is Naja kaoulhia. Figure 4L is Naja alra. Figure 4M is Naja naja) rescued by heparin. Figures 4N to 40 are graphs showing that no rescue is seen in more distantly related snakes (Figure 4N is Echis ocellatus and Figure 40 is Bitis arietans). Significance determined by simple linear regression.

[0019] Figure 5 illustrates that snake venom induced dermonecrosis is inhibited by heparinoids in vivo. Figure 5A is a graph depicting MTT cell viability and PI cell death assays on HaCaT epidermal keratinocytes exposed to serial dilutions (4.74-47.4 pg / mL) of spitting cobra venoms. Figure 5B is a graph depicting MTT-quantified %-cell viability and Figure 5C is a graph depicting Pl-quantified %-cell death of HaCaT keratinocytes treated with venoms preincubated with saline vehicle control or heparin, dalteparin or tinzaparin (1000 pg / mL). Figure 5D is an illustration depicting Mice ID-inj ection with venom that had been pre-incubated with saline vehicle control, dalteparin or tinzaparin (60 pg [3 mg / mL]). After 72 h mice were euthanized and the internal skin lesions excised for photographs and height and width measurements with calipers, from which area was calculated (bar graphs represent the mean lesion area for each treatment group and error bars represent SEM). Figure 5E is representative images of necrosis, scale bar = 5 mm. Figures 5F to 5H are calculated areas for treatments using 25 pg N pallida venom (in Figure 5F), 57 pg Nigerian N. nigricollis venom (in Figure 5G), or 63 pg Tanzanian N. nigricollis venom (in Figure 5H), n>4 (significance was determined by One-way ANOVA and Dunnett test, *P<0.05, **P<0.01). Figure 51 is an illustration depicting post-envenoming treatment. Figures 5 J and 5K depict effect of ID-inj ection with Tanzanian N. nigricollis venom (63 pg) immediately followed by ID-inj ection at the venom injection site with saline vehicle control, low dose (3 mg / kg) or moderate ‘human-equivalent’ dose (21.5 mg / kg) tinzaparin, and Figures 5L and 5M depict effect of ID-inj ection with Tanzanian TV. nigricollis venom (63 pg) immediately followed by SC-inj ection underneath the venom injection site with saline vehicle control, low dose (3 mg / kg) or moderate ‘human-equivalent’ dose (21.5 mg / kg) tinzaparin.Figure 5N is light micrograph images (100X) of hematoxylin & eosin (H&E)-stained skin lesion cross-sections from mice injected with Tanzanian N. nigricollis venom (63 pg) and preincubated saline control show severe damage, with complete loss of the epidermis and necrosis of the underlying dermis, hypodermis, and panni cuius camosus. Sections from mice injected with pre-incubated tinzaparin (3 mg / kg) or receiving SC-injected tinzaparin (21.5 mg / kg) immediately post-envenoming show only minimal damage, with mild reactive changes including epidermal hyperplasia and dermal inflammation.

[0020] Figure 6 illustrates CRISPR KO screen additional information. Figure 6A is a graph depicting inhibition of apoptosis (Ac-DEVD-CHO; Z-VAD-FMK), necroptosis (NSA, Nec-1), or a combination on HAP1 cells treated with spitting cobra venom (10 pg / mL). Figure 6B is a graph depicting performance in N. pallida screen of example sgRNAs. Top 3 sgRNAs targeting sensitizing genes, Top 15 sgRNAs targeting resistance genes and control. Figure 6C is a graph depicting performance in N. nigricollis screen of example sgRNAs. Top 3 sgRNAs targeting sensitizing genes, Top 15 sgRNAs targeting resistance genes and control. Figure 6D is rank plot showing log2(fold changes) (LFCs) in the N pallida screen. Figure 6E is rank plot showing log2(fold changes) (LFCs) in the TV. pallida screen.

[0021] Figure 7 is a graph illustrating Tanzanian N. nigricollis venom cytotoxicity. HAP1 cell viability is determined by resazurin assays after 24 h treatment with serial dilutions of Tanzanian N. nigricollis venom (n = 3).

[0022] Figure 8 illustrates additional flow cytometry information. Figures 8A to 8C are representative flow cytometry histograms of WT HAP1 in gray and cells exposed to Alexa488- tagged Nigerian N. nigricollis venom only (Figure 8A), venom with heparin (Figure 8B), or venom with tinzaparin (Figure 8C). Figure 8D is a graph depicting quantification of binding intensity (n = 5). Significance was determined by One-way ANOVA and Dunnett test, **P<0.01, ****p<0.0001. Figures 8E to 8G are representative flow cytometry histograms of WT HAP1 in gray and cells exposed to Alexa488-tagged Tanzanian TV. nigricollis venom only (Figure 8E), venom with heparin (Figure 8F), or venom with tinzaparin (Figure 8G). Figure 8H is a graph depicting quantification of binding intensity (n = 5). Significance was determined by One-way ANOVA and Dunnett test, *P<0.05.

[0023] Figure 9 illustrates Tanzanian N. nigricollis venom binding. Figure 9A is a graph depicting heparin affinity chromatography of Tanzanian TV. nigricollis venom. Unbound (U), Peak 2 (P2) and Peak 3 (P3). Figure 9B is a graph depicting cation exchange chromatography of Peak 3. Figure 9C is a photo of SDS-PAGE gel of whole venom and resulting toxin fractions. Figure 9D is surface plasmon resonance (SPR) results. Representative normalized sensorgrams of toxin binding to tinzaparin. Figure 9E is fits of the SPR data from Figure 9D to a 1 : 1 binding model are shown. KDS are indicated on each plot. Figure 9F is graphs depicting cytotoxicity of lOpg / mL of each toxin fraction and rescue by 20pM tinzaparin. Significance determined by 2- way ANOVA and Sydak test, **P<0.01, ****P<0.0001 (n = 3).

[0024] Figure 10 illustrates Nigerian TV. nigricollis venom binding. Figure 10A is a graph depicting heparin affinity chromatography of Nigerian TV. nigricollis venom. Unbound (U), Peak 2 (P2), Peak 3 (P3) and Peak 4 (P4). Figure 10B is a photo of SDS-PAGE gel of whole venom and resulting toxin fractions. Figure 10C is surface plasmon resonance (SPR) results. Representative normalized sensorgrams of toxin binding to tinzaparin. Figure 10D is fits of the SPR data from Figure 10C to a 1 : 1 binding model are shown. KDS are indicated on each plot. (E) Cytotoxicity of 10 pg / mL of each toxin fraction and rescue by 20 pM tinzaparin. Significance determined by 2-way ANOVA and Sydak test, ***P<0.001, ****P<0.0001 (n = 3).

[0025] Figure 11 illustrates heparin and dalteparin binding to TV. pallida toxins. Figure 11A is surface plasmon resonance (SPR) results. Representative normalized sensorgrams of TV. pallida toxin binding to heparin. Figure 11B is fits of the SPR data from Figure 11A to a 1 : 1 binding model are shown. KDS are indicated on each plot. Figure 11C is representative normalized sensorgrams of TV. pallida toxin binding to dalteparin. Figure 11D is fits of the SPR data from (C) to a 1 : 1 binding model are shown. KDS are indicated on each plot.

[0026] Figure 12 depicts all lesion images from every mouse in pre-incubation trials, minus those that were culled before the desired timepoints due to humane endpoints being reached. Mice were ID injected in the shaved rear quadrant on the dorsal side of the flank skin with TV. pallida (25 pg), Nigerian TV. nigricollis (57 pg), or Tanzanian TV. nigricollis (63 pg) venom that had been pre-incubated with drug vehicle control, dalteparin or tinzaparin (60 pg [3 mg / mL]). After 72 hours the mice were euthanized and their lesions excised, measured with calipers, and photographed. Scale bar = 5 mm.

[0027] Figure 13 depicts all lesion images from every mouse in post-envenomation trials, minus those that were culled before the desired timepoints due to humane endpoints being reached. Figure 13A depicts mice that were ID injected in the shaved rear quadrant on the dorsal side of the flank skin with Tanzanian N. nigricollis (110 pg) venom immediately followed by ID injection of drug vehicle, low dose (3 mg / kg) or moderate ‘human-equivalent’ dose (21.5 mg / kg) tinzaparin. Figure 13B depicts mice that were ID injected in the shaved rear quadrant on the dorsal side of the flank skin with Tanzanian N. nigricollis (110 pg) venom immediately followed by SC injection of drug vehicle, low dose (3 mg / kg) or moderate ‘human-equivalent’ dose (21.5 mg / kg) tinzaparin. After 72 hours the mice were euthanized and their lesions excised, measured with calipers, and photographed. Scale bar = 5 mm. * This data point was formally identified as an outlier based on a Grubb’s outlier test (Alpha = 0.2).

[0028] Figure 14A depicts result of gene enrichment analysis of screens performed using MAGeCK. Horizontal dotted line indicates -logio(false discovery rate) (FDR) = 1 and vertical dotted lines indicate log2(fold changes) (LFCs) of -2 and 2. Plots generated usingEnhanced Volcano (vl.10.0) R package. Genes within proteoglycan biosynthesis pathway highlighted in blue. Figure 14B depicts a graph showing HAP1 cell viability as determined by resazurin assays after 3 day treatment with sea nettle venom 20pg / mL, and serial dilutions of heparan sulfate, heparin or chondroitin sulfate. Figure 14C depicts a graph showing HAP1 cell viability as determined by resazurin assays after 3 day treatment with sea nettle venom 20pg / mL, and serial dilutions of heparan sulfate, heparin or chondroitin sulfate. Figures 14D and 14E show that CRISPR knockout of heparan sulfate biosynthesis genes confers protection against sea nettle venom (SNV) cytotoxicity. Figure 14D depicts a schematic representation of the heparan sulfate biosynthesis pathway. Gene products involved in the pathway are listed (gene names with single underline indicates CRISPR ko screen hits and gene names with dashed underline indicates proteins not identified in the screen). Figure 14E depicts bar graphs showing pools of single sgRNA knockout cells for non-targeting control (NTC), a negative on-target control (TMEM50A, referred to as NEG) and heparan sulfate biosynthesis gene hits were generated by lentiviral transduction, with KO scores of cell pools determined by Synthego ICE analysis. Topranked sgRNAs for pathway hits XYLT2, B4GALT7, B3GAT3, EXTL3, EXT1, EXT2 and SLC35B2 were cloned into lentiCRISPRv2 and used for validation studies. Wild-type (WT), NTC and KO cells were treated with 30 pg / mL SNV for 24 hours before viability was determined by resazurin assay, normalized against untreated conditions for each pool. Significance was determined using an unpaired one-tailed parametric t-test with Welch’scorrection; *P < 0.05, **P < 0.01, ***p < 0.001, and ****p < 0.0001 (n = 4). Figures 14F to 141 illustrate that sea nettle venom induced pain is rescued by heparin in vivo. Figure 14F depicts a schematic of pain behaviour assays. Baseline behaviour was recorded one week prior to experimental conditions. Mice were injected intraplantar with either Sea Nettle venom (50 pg) only, venom (50 pg) preincubated with heparin (60 pg) for 30 min, or HEPES buffer control. Mice then underwent a battery of pain behavioural assays. Figure 14G depicts a graph showing the result of spontaneous pain assay. Paw licking was recorded for first 10 mins post injection. Figure 14H depicts a graph showing the result of Hargreaves assay. Hargreaves heat sensitisation was at 1 and 6 h post injection. Figure 141 depicts a graph showing the result of Von Frey assay. Von Frey mechanical sensitisation was at 7 h post injection. Significance was determined by 2-way ANOVA and Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***p < 0.001, and ****p < 0.0001 (n as stated in graph).

[0029] Figure 15A depicts a graph showing HAP1 cell viability as determined by resazurin assays after 3 day treatment with serial dilutions of blue bottle venom (n = 3). Figures 15B and 15C depicts result of gene enrichment analysis of screens performed using MAGeCK. Horizontal dotted line indicates -logio(false discovery rate) (FDR) = 1 and vertical dotted lines indicate log2(fold changes) (LFCs) of -1 and 1. Plots generated using EnhancedVolcano (vl.10.0) R package. Genes within proteoglycan biosynthesis pathway highlighted in blue (Figure 15B). Genes within GPI anchor biosynthesis pathway highlighted in red (Figure 15C). Figure 15D depicts graphs showing bluebottle venom (2.5 mg / mL) and serial dilutions of heparin, tinzaparin and dalteparin (10-40 pM) were added simultaneously to HAP1 cells. Resazurin cell viability assays were performed after 24 h of treatment. Significance determined by 2-way ANOVA and Dunnett test,**P<0.01, ***P<0.001, ****P<0.0001 (n = 3). Figures 15E and 15F shows that heparan sulfate biosynthesis is required for bluebottle venom cytotoxicity. Figure 15E depicts a schematic representation of heparan sulfate biosynthesis pathway. Figure 15F depicts bar graphs showing pools of knockout cells (right bar on each graph) for selected hits and non targeting control guide (left baf on each graph) are compared. sgRNA KO provides protection from venom. Cells are treated with 2.5 mg / mL P. utriculus venom for 24 h and viability determined by resazurin. Significance was determined by the one-tailed Mann-Whitney test; *P < 0.05 (n =3). Figures 15G to 15J illustrate that bluebottle venom induced pain is rescued by heparin in vivo. Figure 15G depicts a schematic of pain behaviour assays. Baseline behaviour was recorded one week prior to experimental conditions. Mice were injected intraplantar with either P. utriculus venom (500 pg) only, venom (500 pg) preincubated withheparin (60 pg) for 30 min, or HEPES buffer control. Mice then underwent a battery of pain behavioural assays. Figure 15H depicts a graph showing the result of spontaneous pain assay. Paw licking was recorded for first 10 mins post injection. Figure 151 depicts a graph showing the result of Hargreaves assay. Hargreaves heat sensitisation was at 1, 6 and 24 h post injection. Figure 15 J depicts a graph showing the result of Von Frey assay. Von Frey mechanical sensitisation was at 7 h post injection. Significance was determined by 2-way ANOVA and Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***p < 0.001, and ****p < 0.0001 (n as stated in graph).

[0030] Figure 16A depicts a graph showing HAP1 cell viability as determined by resazurin assays after 3 day treatment with serial dilutions of Assassin bug venom( / / = 3) is rescued by heparin. Figure 16B depicts a graph showing HAP1 cell viability as determined by resazurin assays after 3 day treatment with serial dilutions ofU. ferox toxin (n = 3) is rescued by heparin.Description

[0031] Definitions

[0032] Definitions of common terms in cellular and molecular biology, and biochemistry can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 9780911910421, 0911910425); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 2008 (ISBN 3527305424, 9783527305421); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1- 56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2016 (ISBN 9780815345510, 0815345518); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al , Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Laboratory Methods in Enzymology: RNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN: 9780124200371, 0124200370); Current Protocols in Molecular Biology(CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), Immunological Methods, Ivan Lefkovits, Benvenuto Pemis, (eds.) Elsevier Science, 2014 (ISBN: 9781483269993, 148326999X), the contents of which are all incorporated by reference herein in their entireties.

[0033] As used in this specification and the appended claims, terms in the singular and the singular forms “a,” “an” and “the,” for example, optionally include plural referents unless the content clearly dictates otherwise. For example, “a” venom includes one venom, one or more venoms and a plurality of venoms.

[0034] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0035] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0036] The term “about” as used herein contemplates a range of values for a given number of ±25% the magnitude of that number. In other embodiments, the term “about” contemplates a range of values for a given number of ±30%, ±20%, ±15%, ±10%, or ±5% the magnitude of that number. For example, in one embodiment, “about 60 minutes” indicates a value of 54 to 66 minutes (i.e. 60 minutes ±10%), and the like.

[0037] Numeric ranges are inclusive of the numbers defining the range. It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein.Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0038] The headings provided herein are not intended to limit the disclosure.

[0039] The terms “to treat”, “treating” and “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of venom-induced injury including local tissue damage and / or skin irritation such as dermonecrosis, diminishment of the extent of the disease manifestations, stabilized (i.e. not worsening) state of a symptom or manifestation, “To treat”, “treating” and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0040] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0041] As used herein, the term “venom” or “toxin” refers to a whole venom or toxin obtained from an organism, one or more fractions of venom or toxin from one organism, one or more components from one venom or toxin, or combinations thereof. The fractions may be obtainable by any fractionation method known in the art. The components may be crude, purified or modified. The components may be obtained from natural sources (including gene modified cells, e.g., bacteria) or they may be synthesized chemically.

[0042] As used herein, the term “dermonecrosis” refers a form of injury to the skin cells which results in the premature death of the skin cells. Accordingly, the terms “venom induced dermonecrosis” means that the dermonecrosis is caused by or resulting from exposure of the skin cells to the venom.

[0043] The term “administered” as used herein means administration of an effective amount of one or more heparin and / or heparinoids to a cell either in cell culture or in a subject.

[0044] The term “subject” as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans, companion animals (e.g. dogs, cats, rodents, rabbits etc.) and livestock (e.g. cattle, sheep, pigs, goats, equines such as horses, mules and donkeys etc.).

[0045] As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject.

[0046] As used herein, the terms “effective amount” or “therapeutically effective amount” and the like means an amount effective, at dosages and for periods of time necessary to achieve a desired result. Effective amounts may vary according to factors such as the age, sex, weight and / or species of the subject. The amount of heparin and / or heparinoids that will correspond to such an amount will vary depending upon various factors, such as the pharmaceutical formulation, the route of administration, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

[0047] As used herein, the term “heparin” refers to polyanionic polysaccharides, consisting of alternating uronic acids and glucosamines, with molecular weights of 6,000 to 30,000 g / mol. These polysaccharides are naturally occurring and artificially obtained disaccharide derivatives, such as glucosamine-N-sulfate-, glucosamine-O-sulfate-, glucuronacid-O-sulfate mucopolysaccharides or repeating disaccharides exhibiting properties related to those of heparin. Further comprised is natural, unfractionated heparin, artificial heparin as well as processed heparin as e.g. low molecular weight heparin (LMWH), which has a lower molecular weight than unfractionated heparin, naturally occurring and half or fully synthesized heparinoids and mucopolysaccharides that act similar to heparin.

[0048] As used herein, term “heparinoids” refers to glycosaminoglycans which are chemically and pharmacologically related to heparin. The terms “low molecular weight heparin” and “low molecular weight heparinoids” are used interchangeably and they refer to heparinoid derived from unfractionated heparin by digestion or depolymerization of longer chains of heparin into shorter chains by chemical or enzymatic means. The size of low molecular weight heparinoids is variable but most lie within 3000 to 6000 Da, with chains of mean molecular weight of 5000.Examples of low molecular weight heparinoids include but are not limited to tinzaparin, dalteparin, enoxaparin, and the like.

[0049] As used herein, the term “three-finger toxins” or “3FTxs” refers to a protein superfamily of small toxin proteins typically found in the venoms such as venoms of snakes. They have a common structure of three beta-stranded loops extending from a central core containing all four conserved disulphide bonds. Examples of three-finger toxins include but are not limited to 3FTx cytotoxins CTx3 (cardiotoxin 3), 3FTx cytotoxins CTx4 (cardiotoxin 4), and the like.

[0050] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination. Any example or embodiment herein shall be taken to apply mutatis mutandis to any other example or embodiment unless specifically stated otherwise.

[0051] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent methods and systems are clearly within the scope of the disclosure, as described herein.

[0052] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group ofcompositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0053] The disclosure is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying drawings. Although the examples herein concern humans and the language is primarily directed to human concerns, the concepts described herein are applicable to other animals. These and other aspects and features of the present disclosure will become more fully apparent from the following description and appended claims, or may be learned by the practice of the disclosure as set forth hereinafter.

[0054] A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

[0055] Overview

[0056] Using a functional genomics approach, the inventors of the present application identify human genes that genetically interact with venoms from various species of animals and plants, such as venoms from siphonophores, jellyfish, insects, cobras, and plants. The inventors surprisingly found that genes conferring resistance to venom cytotoxicity typically control proteoglycan biosynthesis and thus heparin and / or heparinoids can be used as inhibitors of those venoms. In the case of cobra venom, the inventors also surprisingly found that heparinoids prevent venom cytotoxicity by inhibiting three finger cytotoxins. Accordingly, the inventors considers that heparin and / or heparinoid can be used for treating or preventing venom induced injury including local tissue damage and / or skin irritation.

[0057] Thus, the present invention provides a method of treating or preventing venom induced injury including local tissue damage and / or skin irritation which comprises administering to a subject a composition comprising an effective amount of heparin and / or heparinoid. The present invention also provides use of a composition comprising an effective amount of heparin and / or heparinoid in the manufacture of a medicament for treating or preventing venom induced injury including local tissue damage and / or skin irritation. The present invention also provides a composition comprising an effective amount of heparin and / or heparinoid for use in treating or preventing venom induced injury including local tissue damage and / or skin irritation.

[0058] In one embodiment, the venom comprises three-finger toxins (3FTxs). In another embodiment, the venom comprises one or more other toxin. In yet another embodiment, the venom comprises three-finger toxins (3FTxs) and one or more other toxin.

[0059] In one embodiment, the three-finger toxins (3FTxs) comprised in the venom are three finger toxin cytotoxins. A skilled person is aware that three finger toxin cytotoxins are sometime referred to as cardiotoxins. In another embodiment, the three-finger toxins (3FTxs) comprised in the venom are 3FTx cytotoxins CTx3. In another embodiment, the three-finger toxins (3FTxs) comprised in the venom are 3FTx cytotoxins CTx4. In yet another embodiment, the three-finger toxins (3FTxs) comprised in the venom are 3FTx cytotoxins CTx3 and 3FTx cytotoxins CTx4.

[0060] In one embodiment, the other toxin comprised in the venom are toxins that interact with or stick to heparin. In another embodiment, the other toxin comprised in the venom are toxins that interact with or stick to heparin. In yet another embodiment, the other toxin comprised in the venom are toxins that interact with or stick to heparin and heparinoids.

[0061] In one embodiment, the heparinoid used is a low molecular weight (LMW) heparinoid. In one embodiment, the LMW heparinoids is a tinzaparin. In another embodiment, the LMW heparinoids is a dalteparin. In another embodiment, the LMW heparinoids is an enoxaparin. In yet another embodiment, the LMW heparinoids is a pharmaceutically acceptable salt of tinzaparin. In yet another embodiment, the LMW heparinoids is a pharmaceutically acceptable salt of dalteparin. In yet another embodiment, the LMW heparinoids is a pharmaceutically acceptable salt of enoxaparin.

[0062] In one embodiment, the composition comprising heparin and / or heparinoid is administered to a skin of the subject. A person skilled in the art is aware of the typical methods used for administration of the composition comprising heparin and / or heparinoid to a skin of the subject. In one embodiment, the composition comprising heparin and / or heparinoid is administered topically. In another embodiment, the composition comprising heparin and / or heparinoid is administered subcutaneously. In yet another embodiment, the composition comprising heparin and / or heparinoid is administered transcutaneously.

[0063] In one embodiment, the composition comprising heparin and / or heparinoid is administered in less than 5 minutes after exposure to the venom. In another embodiment, the composition comprising heparin and / or heparinoid is administered in less than 10 minutes afterexposure to the venom. In yet another embodiment, the composition comprising heparin and / or heparinoid is administered in less than 30 minutes after exposure to the venom. In yet another embodiment, the composition comprising heparin and / or heparinoid is administered in less than 60 minutes after exposure to the venom. In yet another embodiment, the composition comprising heparin and / or heparinoid is administered about 60 minutes after exposure to the venom.

[0064] A skilled person is aware that the composition comprising the heparin and / or heparinoid can be provided as any type of formulations useful for treating or preventing venom induced injury including local tissue damage and / or skin irritation. A skilled person is also aware that as the composition is also meant to prevent venom-induced injury, the formulation may be a prophylaxis. In one embodiment, the composition comprising heparin and / or heparinoid is provided as a topical formulation. A skilled person is aware that the topical formulation can be of any type including, but is not limited to, cream, ointment, paste, lotion, gel, and the like. In another embodiment, the composition comprising heparin and / or heparinoid is provided as a topical cream formulation. In yet another embodiment, the composition comprising heparin and / or heparinoid is provided as a spray formulation. In yet another embodiment, the composition comprising heparin and / or heparinoid is provided as an injectable formulation. In yet another embodiment, the composition comprising heparin and / or heparinoid is provided as a sunscreen formulation. A skilled person is aware that some of the formulations listed herein (such as sunscreen) may be used as a prophylactic. For example, the sunscreen may be applied before the subject is exposed to the venom.

[0065] A skilled person is aware that local tissue damage, the skin irritation, or the local tissue damage and skin irritation can be of any types provided that they are induced by venoms. In one embodiment, the venom induced local tissue damage and / or skin irritation includes but is not limited to dermonecrosis and the like.

[0066] A skilled person is aware that the composition comprising an effective amount of heparin and / or heparinoid is effective against any venoms comprising three-finger toxins (3FTxs) and / or one or more other toxin originating from various types of plants and animals. In one embodiment, the venom treated by the composition comprising heparin and / or heparinoid originates from a siphonophore. In a preferred embodiment, the siphonophore or jellyfish like animals include but are not limited to Physalia utriculus (blue bottle) Physalia spp., and the like. In another embodiment, the venom treated by the composition comprising heparin and / orheparinoid originates from a jellyfish. In a preferred embodiment, the jellyfish include but are not limited to Chrysaora quinquecirrha (sea nettle) and the like. In yet another embodiment, the venom treated by the composition comprising heparin and / or heparinoid originates from an insect. In a preferred embodiment, the insects include but are not limited to Pristhesancus plagipennis (assassin bug), and the like. In yet another embodiment, the venom treated by the composition comprising heparin and / or heparinoid originates from a plant. In a preferred embodiment, the plants include but are not limited to Urtica ferox (New Zealand stinging nettle), and the like. In yet another embodiment, the venom treated by the composition comprising heparin and / or heparinoid originates from a snake. In a preferred embodiment, the snake is a cobra. In a more preferred embodiment, the cobras include but are not limited to Nigerian Naja nigricollis (black-necked spitting cobra), Tanzanian Naja nigricollis (black-necked spitting cobra), Naja pallida (red-necked spitting cobra), Naja kaouthia (monocled cobra), Naja atra (Chinese cobra), Naja naja (Indian spectacled cobra), and the like.

[0067] Examples

[0068] 1, Example 1

[0069] Venoms across varying taxa were tested to see the broad applicability of heparin and heparinoids. The venoms discussed in Example 1 do not contain cytotoxic three-finger toxins, but they are able to interact with and / or stick to heparin and / or heparinoids. Presently, the precise mechanisms and toxin components that the heparin or heparinoids are acting against have not been elucidated.

[0070] 1, 1, Effect of Heparin and LMW Heparinoids on Blue Bottle (Physalia spp., Physalia iilriciiliis } Venom

[0071] To determine the cytotoxicity of blue bottle venom, HAP1 cells were trypsinized and seeded in 96 well plates at a density of 3.5 xlO4cells / well. After 24 h, serial dilutions of bluebottle venom were added for a further 3 days. After incubation resazurin solution was added to a final concentration of 30 pg / mL and incubated for 2.5-3 h at 37 °C. The fluorescence was measured at 544 nm excitation and 590 nm emission using a microplate spectrophotometer (FLUOstar Omega, BMG Labtech). As depicted in Fig. 15A, blue bottle venom is cytotoxic to human cells.

[0072] The inventors conducted whole genome CRISPR KnockOut (KO) screening against blue bottle venom using similar methodology further discussed in Example 2. Result of this screening is depicted on Figs. 15B and 15C. The significant genes that promoted venom resistance when targeted included multiple components of the proteoglycan biosynthesis machinery including XYLT2, GPC3, NDST1, EXT1, and B4GALT5 and multiple components involved in biosynthesis and attachment of Glycosylphosphatidylinositols (GPI) anchor including PIGM, PIGC, GPAA1, PIGV, DPMI, UNC50, PIGT, PIGF, PIGB, PIGP, PIGW, PGAP2, and PIGL.

[0073] To validate the pathway hits of the screen, the inventors generated single KO cell pools with sgRNAs that targeted each resistance gene individually and tested changes in cytotoxicity. Two guides were used for each gene hit (XYLT2, EXT1, NDST1 and GPC3; Fig. 15F) and successful KO was evaluated through CRISPR ICE analysis. Targeting of EXT1, NDST1, and GPC3 components of the heparan / heparin biosynthesis pathway conferred some resistance to venom. These data demonstrate that permanent knockout of elements of the heparan sulfate biosynthetic pathway in human cells offers resistance to bluebottle venom, suggesting a role for the heparan sulfate proteoglycan in venom binding and / or uptake at the cell surface.

[0074] The main symptom of envenoming is a painful rash, therefore the inventor moved into a more physiological relevant mouse model to test the ability of heparin to block venom-induced pain. Set up and schematics for these assays are depicted on Fig. 15G. All baseline behaviour was recorded a week before experimental conditions. Mice were injected intraplantar (footpad) with a dose of venom preincubated with HEPES buffer vehicle or heparin. These mice were then placed through a battery of behavioural tests to assess their pain. After injection and recovery from isoflurane anaesthesia, mice were placed in raised Perspex boxes and 10 mins of video was recorded. This was manually scored by a blind investigator for paw licking which is a common measurement for spontaneous pain (result is depicted on Fig. 15H). Following from this and after at least 1 h of habituation, Hargreaves pain assay was administered. This measures sensitivity to thermal stimuli suggesting thermal hyperalgesia as a sigh of increased pain perception (Hargreaves, result is depicted on Fig. 151). Von Frey assays are also performed to measure mechanical allodynia or hyperalgesia where mice display increased sensitivity to normally non-painful mechanical stimuli (result is depicted on Fig. 15J).

[0075] Venom only mice showed significant increase in spontaneous pain when compared to vehicle only whilst those who received venom and heparin showed no significant increase in pain from control. Venom only mice also displayed thermal hyperalgesia at 1 h and 6 h post injection, with recovery seen at 24 h. Although venom plus heparin mice also showed this increase in pain at 1 h, the recovery was much faster and by 6 h were doing as well as the control. However, this is not reflected in the mechanical sensitisation which at 7 h is still prevalent in both venom and venom plus heparin mice. This data suggests that heparin is able to rescue the inflammatory pain caused by the bluebottle venom.

[0076] Considering that proteoglycans are targeted by both cobra venom (as further discussed in Example 2) and by blue bottle venom, the anti-cobra venom drugs are also tested against the blue bottle venom. Briefly, HAP1 cells were trypsinized and seeded in 96 well plates at a density of 3.5 xlO4cells / well. After 24 h, 2.5 mg / mL bluebottle venom was added for a further 24 hrs with heparin, tinzaparin, or dalteparin in varying concentrations (uM). After incubation resazurin solution was added to a final concentration of 30 pg / mL and incubated for 2.5-3 h at 37 °C. The fluorescence was measured at 544 nm excitation and 590 nm emission using a microplate spectrophotometer (FLUOstar Omega, BMG Labtech). Result of this test is depicted on Fig 15D (from left to right) which indicates that I OpM of heparin, dalteparin, or tinzaparin is effective against 2.5 mg of blue bottle venom.

[0077] 1 ,2 Effect of Heparin on Sea Nettle (Chrysaora quinquecirrha} Venom

[0078] The inventors conducted whole genome CRISPR KnockOut (KO) screening against sea nettle venom using similar methodology further discussed in Example 2. Result of this screening is depicted on Fig. 14A. The significant genes that, when targeted, promoted venom resistance included multiple components of the proteoglycan biosynthesis machinery including XYLT2, EXT1, EXT2, EXTL3, B3GAT3, B4GALT7, and SLC35B2.

[0079] The generated single KO HAP1 cell pools with single sgRNAs and evaluated their proposed rescue of cell viability compared to non-targeting control sgRNA (NTC) cell pools. The result of this screening is depicted on Fig. 14E. Knockout of all of the seven tested hits in the pathway (XYLT2, B4GALT7, B3GAT3, EXTL3, EXT1, EXT2, SLC35B2) significantly improved cell viability post-venom challenge.

[0080] Because the genes XYLT2, EXT1, EXT2, EXTL3, B3GAT3, B4GALT7, and SLC35B2 are part of the heparan sulfate biosynthesis pathway but are also part of the broader proteoglycan biosynthesis pathway that encompasses chondroitin sulfate, the inventors also tested the effect of heparin, heparan sulfate, and chondroitin sulfate in blocking cytotoxicity of sea nettle venom. Briefly, HAP1 cells were trypsinized and seeded in 96 well plates at a density of 3.5 xlO4cells / well. After 24 h, 20pg / mL sea nettle venom was added for a further 3 days with heparan sulfate, heparin or chondroitin sulfate in varying concentrations (pM). After incubation resazurin solution was added to a final concentration of 30 pg / mL and incubated for 2.5-3 h at 37 °C. The fluorescence was measured at 544 nm excitation and 590 nm emission using a microplate spectrophotometer (FLUOstar Omega, BMG Labtech). As shown on Fig 14B and 14C, heparin at concentration of 0.94 pM or above is able to block venom cytotoxicity.

[0081] Pain behaviour was assessed as in Example 1.1. above. Set up and schematics for these assays are depicted on Fig. 14F. Venom only individuals showed a trend in increasing spontaneous pain but this seems to be bimodal with many animals not displaying any paw licking behaviour (result is depicted on Fig. 14G). Hargreaves showed that venom only mice were sensitised at 1 hr but recovered by 6h. Remarkably, the mice who were given venom and heparin were significantly less sensitive to the thermal stimuli and not different to controls at 1 h (result is depicted on Fig. 14H). This is also reflected in the Von Frey assay where venom greatly reduced the withdrawal threshold at 7 h, whilst venom plus heparin animals were able to tolerate higher stimulation (result is depicted on Fig. 141). Overall, this is promising data which shows that heparin can be used to prevent the pain associated with sea nettle envenoming.

[0082] 1,3 Effect of Heparin on Assassin Bug (Pristhesancus plagipennis} Venom and New Zealand Stinging Nettle (Urtica ferox} Toxin

[0083] The effects of heparin against venoms from assassin bug and New Zealand stinging nettle were tested. Briefly, HAP1 cells were trypsinized and seeded in 96 well plates at a density of 3.5 xlO4cells / well. After 24 h, serial dilutions of venom / toxins were added for a further 3 days with or without heparin 20pM. After incubation resazurin solution was added to a final concentration of 30 pg / mL and incubated for 2.5-3 h at 37 °C. The fluorescence was measured at 544 nm excitation and 590 nm emission using a microplate spectrophotometer (FLUOstar Omega, BMG Labtech). As depicted in Fig 16A, 20pM of heparin is effective against up to60pg / mL of assassin bug venom. As depicted in Fig 16B, 20pM of heparin is effective against up to IpM of New Zealand stinging nettle toxin.

[0084] 2, Example 2

[0085] 2, 1 , Materials and Methods

[0086] 2, 1,1 Chemicals, Drugs and Biological Materials

[0087] Sydney: Resazurin Sodium Salt (R7017), Iscove’s modified Dulbecco’s medium (IMDM; 13390), heparin sodium (H3393), tinzaparin sodium (T 1490000) and dalteparin sodium (D0070000) were purchased from Sigma-Aldrich (Merck). Dulbecco’s modified Eagle’s medium (DMEM; 11960-044), Puromycin (Al 1138-03), TrypLE Express (12605-028), and penicillin-streptomycin (15140-122) were purchased from Gibco (Thermo Fisher Scientific). Fetal bovine serum (FBS; SFBS-F) was purchased from Bovogen Biologicals.

[0088] Liverpool: Thiazolyl blue methyltetrazolium bromide (MTT; M5655), dimethyl sulfoxide (DMSO; 276855), and propidium iodide (PI; P4170) were purchased from Sigma- Aldrich (Merck). Dulbecco’s modified Eagle’s medium (DMEM; 11574516), fetal bovine serum (FBS; 11573397), FluoroBrite DMEM (A1896701), glutaMAX supplement (35050038), penicillin-streptomycin (11528876), phosphate buffered saline (11503387), and TrypLE Express were purchased from Gibco (Thermo Fisher Scientific). Heparin sodium (H0200000), tinzaparin sodium (T1490000), and dalteparin sodium (D0070000) were purchased from Sigma-Aldrich (Merck). Working stocks were 50 mg / mL in PBS, stored at -20°C and thawed prior to use for experiments.

[0089] 2, 1,2 Venoms

[0090] Venoms were sourced from wild caught or captive bred snakes maintained in the herpetarium of the Liverpool School of Tropical Medicine’s (LSTM) Centre for Snakebite Research Interventions (CSRI; United Kingdom). This facility and its protocols for the husbandry of snakes are approved and inspected by the UK Home Office and the LSTM and University of Liverpool Animal Welfare and Ethical Review Boards. The venom pools were from: East African Naja nigricollis (Tanzanian, TZN), West African Naja nigricollis (Nigerian, NGA), Naja pallida (Tanzanian), captive bred Naja kaoiilhia. Naja alra. and Naja naja, Echisocellatus (Nigerian), and Bitis arietans (Nigerian). Crude venoms were lyophilized and stored at -20 °C. Prior to use, venoms were resuspended to 10 mg / mL in DPBS and stored at -80 °C in small aliquots with freeze-thaw cycles minimized to prevent degradation.

[0091] 2, 1,3 Cell Culture

[0092] HAP1 cells were generously provided by Dr. Thijn R. Brummelkamp. HAP1 and HEK293T cells were cultured in Iscove’s Modified Dulbecco Media (Gibco) and Dulbecco’s Modified Eagle Medium (Sigma- Aldrich) respectively, both containing 10% FBS and IX Penicillin-Streptomycin at 37°C, 5% CO2.

[0093] Immortalized human epidermal keratinocyte cells, HaCaT, were purchased from Caltag Medsystems (Buckingham, UK). Cells were cultured in phenol red-containing DMEM with GlutaMAX supplemented with 10% FBS, 100 ZU / mL penicillin, 250 pg / mL streptomycin, and 2 mM sodium pyruvate (Standard HaCaT medium). For the MTT and PI multiplexed assays, a minimally fluorescent medium was used instead: FluoroBrite DMEM supplemented with 1% GlutaMAX 100X supplement, 1% FBS, 100 lU / mL penicillin, 250 pg / mL streptomycin, and 2 mM sodium pyruvate (Low background fluorescence HaCaT medium). The cells were split and medium changed twice per week up to a maximum of 30 passages. Cells were maintained in a humidified, 95% air / 5% CO2 atmosphere at 37°C (standard conditions).

[0094] 2, 1 ,4 Cell viability and death assays

[0095] HAP1 cells were trypsinized and seeded in 96 well plates at a density of 3.5 xlO4cells / well. After 24 h, serial dilutions of spitting cobra venom (spanning 0.02-50 mg / mL) were added for a further 24 h. After incubation resazurin solution was added to a final concentration of 30 pg / mL and incubated for 2.5-3 h at 37 °C. The fluorescence was measured at 544 nm excitation and 590 nm emission using a microplate spectrophotometer (FLUOstar Omega, BMG Labtech).

[0096] HaCaT cell viability and death were quantified with MTT and PI multiplexed assays, respectively, as previously described in Hall, et al. (S. R. Hall, S. A. Rasmussen, E. Crittenden, C. A. Dawson, K. E. Bartlett, A. P. Westhorpe, L.-O. Albulescu, J. Kool, J. M. Gutierrez, N. R. Casewell, Repurposed drugs and their combinations prevent morbidity-inducing dermonecrosis caused by diverse cytotoxic snake venoms. bioRxiv (2022), p. 2022.05.20.492855.) with thefollowing alterations: On day 1, cells were seeded (10,000 cells / well in half-volume, black-sided, & clear-bottomed 96-well plates [Greiner; 675090]); On day 2, cells were treated (50 pL / well) with: (a) serial dilutions of Nigerian TV. nigricollis, Tanzanian TV. nigricollis, or TV. pallida (4.74- 47.4 pg / mL), or (b) these same three venoms (15 pg / mL) combined with Heparin, dalteparin, or tinzaparin (1,000 pg / mL) in Pl-containing medium for 24 hours. On day 3, PI fluorescence (Ex544 / Emei2) and MTT absorbance (Asso), after 2 h of MTT exposure, were measured on a CLARIOstar Plus Microplate Reader (BMG Labtech).

[0097] 2, 1,5 Lentivirus production

[0098] Toronto KnockOut Library v3 (TKOv3) library plasmid production. HEK293T cells were seeded at 25 xlO6per T-175 flask. Once cells reached 70-90% confluency after 24 h they were transfected using Lipofectamine 3000 (Thermo) with pCAG-VSVG (Addgene plasmid 35616), psPAX2 (Addgene plasmid 12260) and the TKOv3 plasmid library (Addgene plasmid 90294) at a 1 :3:3 ratio. 16 h after transfection the medium was replenished with fresh medium. At 48 h post transfection the lentivirus-containing supernatant was collected and filtered through a 0.45 pm ultra-low protein binding filter (Merck Millipore) and concentrated with 10 000 MW Pierce protein concentrators (ThermoFisher Scientific). The concentrated lentivirus media was aliquoted and stored at -80°C.

[0099] The concentrated lentiviral library was titrated by transducing HAP1 cells with a dilution series of virus supplemented with polybrene (8 pg / mL). After 24 h, the viral medium was replaced with normal medium to allow cell recovery. Medium containing puromycin (1 pg / mL) was added to the transduced cells and selection was conducted for 72 h with puromycin- supplemented medium being refreshed daily. The multiplicity of infection (MOI) of the virus was determined through comparison of percentage of surviving cells to non-infected control cells by resazurin viability assay.[000100] 2, 1,6 Cell transduction using TKO v3 library[000101] Using an MOI of 0.3 as previously titrated, 70 xlO6HAP1 cells were transduced with the TKOv3 lentivirus library with 8 pg / mL of polybrene for ~300-fold library coverage after selection. After 24 h, viral media was refreshed with normal media for cell recovery. At 48 h post transduction, cells were split into two replicates and selected for seven days with medium containing puromycin (1 pg / mL).[000102] 2, 1.7 CRISPR screen for spitting cobra venom modifiers[000103] Pooled library transduced HAP1 cells were split across four T-175 flasks with 19 xlO6cells per flask. After 24 h, medium was replaced with spitting cobra venom containing medium (5 pg / mL) with one flask refreshed with normal medium as a control. Cells were treated for 3 days then allowed to recover in normal media for 1-3 days. Cells were then replated and treated again with venom containing medium and this process was repeated three times with cells collected at 9 days of selection for genomic DNA extraction.[000104] 2, 1,8 Genomic DNA sequencing[000105] Genomic DNA was extracted from cell pellets with the ISOLATE II Genomic DNA Kit (Bioline). Samples were then prepared for Next Generation Sequencing (NGS) via PCR as previously described in Loo, et al. (L. Loo, M. A. Waller, C. L. Moreno, A. J. Cole, A. O. Stella, O.-T. Pop, A.-K. Jochum, O. H. Ali, C. E. Denes, Z. Hamoudi, F. Chung, A. Aggarwal, J. K. K. Low, K. Patel, R. Siddiquee, T. Kang, S. Mathivanan, J. P. Mackay, W. Jochum, L. Flatz, D. Hesselson, S. Turville, G. G. Neely, Fibroblast-expressed LRRC15 is a receptor for SARS-CoV- 2 spike and controls antiviral and antifibrotic transcriptional programs. PLoS Biol. 21, e3001967 (2023)). Samples of gDNA (25 pg for controls and 5 pg for selected samples) were added to NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs) along with the following primers to bind the sgRNA region: NGSCRISPRv2Fl 5’- GGACAGCACAGATCCAGTTTGGT-3’ (SEQ ID NO: 1) and NGSCRISPRv2Rl 5’- GAGCCAATTCCCACTCCTTTCAA-3’ (SEQ ID NO: 2). A second PCR reaction with a staggered primer mix of P5 and P7 indexing primers unique to each sample was prepared as outlined in Joung et al. (J. Joung, S. Konermann, J. S. Gootenberg, O. O. Abudayyeh, R. J. Platt, M. D. Brigham, N. E. Sanjana, F. Zhang, Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat. Protoc. 12, 828-863 (2017).). Reactions were isolated by gel electrophoresis and resultant products sent to NovogeneAIT Genomics Singapore for NGS. Data was analyzed using the MAGeCK (v05.9.2) pipeline to identify genes that sensitize or protect when compared to diversity controls.[000106] 2, 1,9 Ingenuity pathway analysis (IP A)[000107] Ingenuity Pathway Analysis (Qiagen v01-21-03) was utilized to identify the significant biological pathway(s) enriched by the knockout screens. Canonical pathway analysis wasconducted with IPA using a restricted set of significant genes (absolute log2FC > 1 and FDR of <0.1).[000108] 2, 1.10 Generation of single KO cells and gene validation[000109] To validate candidate genes from the screen, top sgRNAs from the parent library were selected and cloned by traditional restriction digestion cloning into pLentiCRISPRv2 (Addgene plasmid 52961) in parallel to a non-targeting control sgRNA. Lentiviruses carrying these sgRNAs were produced as above, and transduced HAP1 cells were selected with 2 pg / mL puromycin for three days. Cells were allowed to recover for a week before analysis. Successful knockout was confirmed by PCR amplification and Sanger sequencing of each sgRNA target site and comparison to wild type through Synthego ICE. sgRNA and PCR / sequencing primer sequences are as follows:[000110] 2, 1,11 Isolation of Heparin Binding Venom Components[000111] Heparin Affinity Chromatography. To isolate heparin binding components of the venoms, crude N. pallida, TZN N. nigricollis, and NGA N. nigricollis venoms were fractionated using heparin affinity chromatography. Initially, crude venoms were dissolved in 0. IX PBS at 1 mg / mL and loaded onto a 5 mL heparin column (HiTrap Heparin HP, Cytiva). Toxins were eluted with a 0-1.2 M NaCl gradient in PBS. Fractionation was assessed by SDS-PAGE and concentration was estimated from absorbance at 280 nm on a NanoDrop (ThermoFisher Scientific). Protein aliquots were snap frozen in liquid nitrogen and stored at -80 °C until required.[000112] Cation Exchange. The major venom constituents of N. pallida and TZN N. nigricollis were further isolated using cation exchange chromatography. P3 of each venom was dialysed against 50 mM sodium phosphate, pH 6 and loaded onto a 1 mL cation exchange column (HiTrap SP HP, Cytiva). The toxins were then separated with a 0-0.6 M gradient of NaCl in 50 mM sodium phosphate, pH 6. 3FTx and bPLA2 eluted in that order. Venom components were dialysed in PBS and used for further assays and taken for mass spectrometry, or in 20 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween 20 for surface plasmon resonance. Protein purification was analyzed by SDS-PAGE and measured via NanoDrop (ThermoFisher Scientific) and Qubit (ThermoFisher Scientific). Protein aliquots were snap frozen in liquid nitrogen and stored at -80 °C until required.[000113] Additional isolation of 3FTx cytotoxins 3 and 4, 20 mg of venom was dissolved in 2 mL 50 mM sodium phosphate, pH 6.0 and then applied to a 4.7 mL HPSP cation exchange chromatography column (Cytiva) equilibrated in the same buffer. Elution was carried out using a 15-column volume (CV) gradient of 0 - 0.7 M NaCl in 50 mM sodium phosphate, pH 6.0. The flow rate was 0.5 mL / min and 1 mL fractions were collected from the start of the NaCl gradient. The peak containing cytotoxins 3 and 4 was made up to 1.2 M in NaCl and loaded onto a 1 mL Phenyl Superose hydrophobic interaction chromatography column. Proteins were then eluted in a 5 CV gradient of 1.2 M NaCl in 25 mM sodium phosphate pH 7.2 to 30% (v / v) ethylene glycol in 25 mM sodium phosphate pH 7.2. The flow rate was 0.5 mL / min and elution was monitored at 214 nm. RP-HPLC and SDS-PAGE analysis showed that the eluted cytotoxins 3 and 4 were pure and were used in the present study after dialysis into PBS.[000114] 2, 1,12 Mass spectrometry[000115] ID liquid chromatography-mass spectrometry (LC-MS) was carried out on an Oritrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Scientific). Raw data were processed and protein sequences identified using Mascot and database derived from Kazandjian, et al. (T. D. Kazandjian, D. Petras, S. D. Robinson, J. van Thiel, H. W. Greene, K. Arbuckle, A. Barlow, D. A. Carter, R. M. Wouters, G. Whiteley, S. C. Wagstaff, A. S. Arias, L.-O. Albulescu, A. Plettenberg Laing, C. Hall, A. Heap, S. Penrhyn-Lowe, C. V. McCabe, S. Ainsworth, R. R. da Silva, P. C. Dorrestein, M. K. Richardson, J. M. Gutierrez, J. J. Calvete, R. A. Harrison, I. Vetter, E. a. B. Undheim, W. Wilster, N. R. Casewell, Convergent evolution of pain-inducing defensive venom components in spitting cobras. Science. 371, 386-390 (2021).).[000116] 2, 1,13 Surface plasmon resonance (SPR)[000117] Heparin, tinzaparin and dalteparin were biotinylated by reaction of EZ-link NHS-LC- LC-Biotin with the free amino groups of unsubstituted heparin glucosamine residues. 100 pM of heparin and heparinoids were prepared in 0.1 M sodium bicarbonate pH 8.5. A five-fold excess of NHS-LCLC-Biotin (570 g / mol in 100% DMSO) was added and the reaction was incubated on ice for 3 h. To remove unreacted biotin, the mixtures was dialysed in 20 mM HEPES pH 7.5 and 150 mM NaCl. Biotinylated heparins were snap-frozen and stored at -80 °C until required.[000118] SPR measurements were taken using a BIAcore T200 instrument (Cytiva) and analyzed using the BIAcore Evaluation software (v3.2). Experiments were performed at 25 °C using the multi cycle kinetics mode. Biotinylated heparins were immobilized onto a CM5 chip (Cytiva) amine-coupled with streptavidin at around 100 response units (RU). 20 mM HEPES pH 7.5, 150 mM NaC and 0.05% Tween-20 was used as the running buffer. Regeneration of the chip with 2M NaCl PBS followed each venom / fraction injection. Non-specific binding of toxins to heparins was determined by injecting the toxins over a control flow cell with streptavidin prepared without bound heparins. Binding to this surface was subtracted in all binding curves. Affinities measured for protein mixtures are concentration weighted means.[000119] 2, 1,14 Conjugation of venom with fluorophores[000120] Whole venoms were conjugated to Alexa Fluor™ 488 as per the protein labeling kit manufacturer’s instructions (Invitrogen). Briefly, 50 pl of 1 M sodium bicarbonate was added to 500 pl of 2 mg / mL crude venom. The solution was then added to Alexa Fluor™ 488 reactive dye and stirred for 1 h at room temperature. Conjugated venom was then loaded into Zeba™ Dye andBiotin Removal Spin Columns and eluted via centrifugation. Tagging efficiency and protein concentrations were determined via NanoDrop (ThermoFisher Scientific).[000121] 2, 1,15 Flow cytometry[000122] Cell surface attachment of tagged venoms was detected by flow cytometry. Briefly, HAP1 cells were incubated with TrypLE at 37 °C and neutralized with IMDM once dissociated.5 xlO5cells were collected and incubated with Alexa Fluor™ 488-conjugated venom (100 pM / mL) with and without heparin (20 pM) or tinzaparin (20 pM) for 30 min on ice. The cells were washed twice with FACS buffer consisting of PBS, 2% BSA and 0.5mM EDTA before resuspending in the same buffer with DAPI (0.1 pg / mL) and analyzed on the Cytek Aurora (Cytek Biosciences).[000123] 2, 1,16 Animal ethics and maintenance[000124] Liverpool, UK: All drug-plus-venom precincubation and ID-envenoming followed by SC-drug dosing animal experiments were conducted using protocols approved by the Animal Welfare and Ethical Review Boards of the Liverpool School of Tropical Medicine and the University of Liverpool and were performed in pathogen-free conditions under licensed approval (PPL #P58464F90) of the UK Home Office and in accordance with the Animal [Scientific Procedures] Act 1986 and institutional guidance on animal care. All experimental animals (18-28 g [4-6 weeks old], male, SWISS (CD-I) mice from Janvier, France or Charles River, UK) were acclimated for a minimum of one week before experimentation with their health monitored daily. Mice were grouped in cages of five, with room conditions of approximately 22 °C at 40-50% humidity, with 12 / 12 hour light cycles, and given ad lib access to CRM irradiated food (Special Diet Services, UK) and reverse osmosis water in an automatic water system. Mice were housed in specific pathogen free facilities in Techniplast GM500 cages containing Lignocell bedding (JRS, Germany), Sizzlenest zigzag fibers as nesting material (RAJA), and supplied with environmental enrichment materials.[000125] San Jose, Costa Rica: All ID-envenoming followed by ID-drug dosing animal experiments were carried out at Institute Clodomiro Picado. These were conducted using protocols approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica (approval number CICUA 82-08). All experimental animals (18-20 g [4-5 weeks old], mixed sex, CD-I mice, Institute Clodomiro Picado, CostaRica) were acclimatized before experimentation. Mice were grouped in cages of five, with room conditions of approximately 22-24 °C at 60-65% humidity, with 12 / 12 hour light cycles, given ad lib access to food and water, and housed in Tecniplast Eurostandard Type II 1264C cages.[000126] 2, 1,17 Preclinical anti -derm onecrosis efficacy of heparinoids via a preincubation model of envenoming[000127] The in vivo experimental design was based upon 3R-refined WHO-recommended envenoming protocols described in Hall, et al. (S. R. Hall, S. A. Rasmussen, E. Crittenden, C. A. Dawson, K. E. Bartlett, A. P. Westhorpe, L.-O. Albulescu, J. Kool, J. M. Gutierrez, N. R. Casewell, Repurposed drugs and their combinations prevent morbidity-inducing dermonecrosis caused by diverse cytotoxic snake venoms. bioRxiv (2022), p. 2022.05.20.492855.), Theakston and Reid (R. D. Theakston, H. A. Reid, Development of simple standard assay procedures for the characterization of snake venom. Bull. World Health Organ. 61, 949-956 (1983).), and Albulescu, et al. (L.-O. Albulescu, M. S. Hale, S. Ainsworth, J. Alsolaiss, E. Crittenden, J. J. Calvete, C. Evans, M. C. Wilkinson, R. A. Harrison, J. Kool, N. R. Casewell, Preclinical validation of a repurposed metal chelator as an early-intervention therapeutic for hemotoxic snakebite. Sci. Transl. Med. 12 (2020), doi: 10.1126 / scitranslmed.aay8314.) The anti- dermonecrosis methods were based on the Minimum Necrotizing Dose (MND) principles originally described in Theakston and Reid. As similar experiments with heparinoids as dermonecrosis treatments have never been performed previously, a priori power calculations were not possible; therefore, groups of 5 mice were randomly allocated into each treatment group based on sample sizes previously used to assess venom inhibition in this model. These groups of five mice (n=5; 45 mice total) received, in random order by a treatment-preparer separate from the treatment-injectors, experimental doses per mouse that consisted of venom from TZN N. pallida (25 pg), TZN N. nigricollis (63 pg), or NGA N. nigricollis (57 pg) combined with saline vehicle control, tinzaparin or dalteparin (60 pg [3 mg / kg]). Albulescu, et al. previously used 60 pg per 20 g mouse (3 mg / kg) of marimastat in their preclinical ID haemotoxicity trials; therefore this same comparative dose was chosen for the heparinoid dermonecrosis trials. In vivo team members were unblinded to the treatment randomization postinjection to allow for the appropriate observation of venom-specific systemic endpoints that would have necessitated the implementation of early euthanasia. Stock solutions of tinzaparin and dalteparin were dissolved in PBS (50 mg / mL) and stored at -20 °C prior to use in these in vivo experiments. All experimental doses were prepared to a volume of 50 pL and preincubatedat 37 °C for 30 minutes the morning of the experiments, then kept on ice for no more than 3 hours until the mice were injected. For dose delivery, mice were briefly anesthetized using inhalational isoflurane (4% for induction of anesthesia, 1.5-2% for maintenance) and ID-injected in the shaved rear quadrant on the dorsal side of the flank skin with the 50 pL treatments. The mice were observed at least three times daily up to 72 hours post-injection to ensure signs of systemic envenoming or excessive external lesion development did not present. At the end of the experiments (72 hours) the mice were euthanized using rising concentrations of CO2, after which the skin surrounding the injection site was dissected and the width and height of internal skin lesions measured with calipers, from which area was calculated, and photographed. Crosssection strips down the middle of the skin lesions were cut with microtome blades, placed in tissue cassettes, and preserved in 10% neutral formalin (BAF-6000-08A; CellPath) before being prepared for downstream histopathological analysis as previously described by Hall, et al.[000128] 2, 1,18 Preclinical anti -derm onecrosis efficacy of small molecule drug combinations via SC and ID ‘rescue’ models of envenoming[000129] SC-drug rescue (UK): Groups of five mice (20 mice total) were ID-injected with TZA N. nigricollis venom (63 pg) diluted in 25 pL of PBS, followed by an immediate 25 pL SC- injection of either: (i) vehicle control (PBS), (ii) 3 mg / kg tinzaparin, or (iii) 21.5 mg / kg of tinzaparin (calculated human equivalent dose based on therapeutic dose of tinzaparin in humans being 175 lU / kg [roughly 1.75 mg / kg]), directly underneath the ID-injected venom. After 72 hours mice were euthanized by CO2 inhalation, the skin surrounding the injection site was dissected, and the internal necrotic lesions were measured and photographed as described above. Separate (n=5) venom -plus-PBS control groups were completed for both the 3 and 21.5 mg / kg tinzaparin trials and data were combined due to being identical treatments.[000130] ID-drug rescue (Costa Rica): Groups of five mice (20 mice total) were pre-treated with the analgesic tramadol (50 mg / kg by the subcutaneous route). Fifteen minutes later, mice were ID-injected with venom from TZA A. nigricollis (63 pg) diluted in 25 pL of PBS, after which they were immediately ID-injected in the same location with 25 pL of tinzaparin vehicle control (PBS) or 3 or 21.5 mg / kg of tinzaparin. After 72 hours mice were euthanized by CO2 inhalation, the skin surrounding the injection site was dissected, and the internal necrotic lesions were measured and photographed as described above. Separate (n=5) venom-plus-PBS control groups were completed for both the 3 and 21.5 mg / kg tinzaparin trials and were combined due to beingidentical treatments. A single lesion measurement from the venom-plus-PBS control group was excluded as only a minor lesion developed in a mouse that the inventors suspect of being misinjected, and thus did not receive the correct venom dose. This data point was formally identified as an outlier based on a Grubb’s outlier test (Alpha = 0.2), but to ensure transparency, images of this lesion are displayed in fig. 13.[000131] 2, 1,19 Data Analysis[000132] Statistical analyses of data, unless otherwise stated, were conducted using GraphPad Prism (9.3.1) software. All error bars in this manuscript report SEM. All analyses were performed at a threshold a level of 0.05. Specific statistical tests used for each dataset are stated in respective figure legends. All flow cytometry data was analyzed using FlowJo Software vl0.6 (BD Life Sciences).[000133] 2,2, Results[000134] 2,2,1 Whole genome CRISPR knockout screens for spitting cobra venom cytotoxicity[000135] One of the main groups of snakes causing death and morbidity worldwide are cobras (Naja spp.). While bites by many cobras cause systemic neurotoxicity, several species, particularly the African spitting cobras (e.g. N. nigricollis, N. pallida, N. mossambica), cause morbidity as the result of severe local envenoming characterised by extensive tissue damage. Venoms from the red (N. pallida, Tanzania) and black-necked (N. nigricollis, Nigeria) spitting cobras (Fig. 1A) showed potent cytotoxicity in the human haploid cell line, HAP1 (Fig. IB). Pharmacological inhibition of apoptosis (Ac-DEVD-CHO; Z-VAD-FMK) did not suppress venom cytotoxicity, however the necroptotic inhibitor necrosulfonamide (NS A) limited some cell death, suggesting cobra venom cytotoxicity may partially trigger necroptotic death (fig. 6A). To guide the development of novel therapeutics, the inventors defined the molecular mechanisms involved in venom induced cell death using whole genome CRISPR KnockOut (KO) screening (Fig. 1C). HAP Is were transduced with the TKOv3 library which targets most human proteincoding genes with ~4 guides / gene. This pool of CRISPR KO cells was then selected with 5 pg / mL of N. pallida or N. nigricollis venom three times. After recovery, guide sgRNA in surviving cells was isolated, amplified by PCR, and quantified by next generation sequencing. Guide enrichment was compared to a control unselected population using the MaGeCK pipeline.Guide RNAs associated with venom sensitization (Log2<-2, FDR<0.1) or resistance (Log2>2, FDR<0.1) were identified, and substantial overlap was observed between the two snake species (Fig. ID and E).[000136] For TV. pallida venom, the top significant genes that, when targeted, promoted venom sensitization include the cell growth / tumour suppressors TSC1 / TSC2, the chromatin remodelling SWI / SNF component SMARCD1, the cyclin dependent kinase CDK13, the histone deacetylase HDAC3, the anti-apoptotic protein ZFAT, and CRAMP1L, an uncharacterized gene linked with susceptibility to skin rash. For A. nigricollis venom, the top sensitizers again included TSC1 / TSC2, the TSC subunit TBC1D7, the SWI / SNF component SMARCC1, the lipid phosphatase Inositol Polyphosphate Phosphatase Like 1 / SHIP2, and the microtubule interaction protein APPBP2.[000137] For A. pallida venom, the top significant genes that, when targeted, promoted venom resistance included the uncharacterized transmembrane protein TMEM50A, the suppressor of growth hormone tetraspanin membrane protein LEPROTL1, and components of proteoglycan biosynthesis NDST1, XYLT2, EXT1, EXTL3, and SLC35B2. For A nigricollis venom, the top promoters again included LEPROTL1 and TMEM50A, as well as multiple components of the proteoglycan biosynthesis machinery including EXT1, B4GALT7, EXT2, EXTL3, XYLT2, NDST1 and SLC35B2. Further, pathway analysis of these data highlighted heparin sulfate, chondroitin sulfate, and dermatan sulfate biosynthesis as critical pathways required for cytotoxicity of both A pallida and A nigricollis venoms (Fig. IF and G).[000138] 2,2,2 Heparin Biosynthesis is required for venom cytotoxicity[000139] The top pathway required for venom cytotoxicity from A pallida or A nigricollis was heparan / heparin sulfate biosynthesis (A pallida'. p<10'10, A nigricollis'. p<10'8) and the screening data showed that targeting of most of the heparan / heparin sulfate biosynthesis pathway components individually was sufficient to block venom activity (A pallida 7 / 11, and A nigricollis 8 / 11 components of the pathway were hit, see Fig. 2A). To validate these results, the inventors targeted each resistance gene individually and tested cytotoxicity. Targeting each component of the heparan / heparin biosynthesis pathway conferred some resistance to each venom (Fig. 2B and 2C), confirming a role for heparan in cobra venom cytotoxicity. To test the generalizability of this requirement, the inventors also treated gene targeted cells with anadditional spitting cobra venom (Fig. 2D, fig. 7; Tanzanian TV. nigricollis), and again components of heparan / h eparin sulfate biosynthesis were required for cytotoxicity.[000140] Heparan and heparin sulfate share a sugar backbone synthesized by a common pathway (Fig. 2A). While heparan sulfate is a ubiquitous component of the extracellular matrix, heparin is primarily produced by tissue mast cells. Heparin is a highly sulfated, polyanionic polysaccharide used clinically for its potent anticoagulant activity. Heparin is on the WHO Model List of Essential Medicines (EML), however, multiple low molecular weight (LMW) medical variants of heparin (tinzaparin, T; dalteparin, D) termed “heparinoids” are also available and approved for antithrombotic use (Fig. 3A). Since heparan / heparin sulfate biosynthesis was necessary for venom to cause cytotoxicity, the inventors hypothesized that adding excess free heparin or LMW heparinoids may be sufficient to block venom cytotoxicity. Indeed, pretreatment with heparin, tinzaparin, or dalteparin, all block cytotoxicity in response to TV. pallida (Fig. 3B, quantified in C), Nigerian TV. nigricollis (Fig. 3B, quantified in D), or Tanzanian TV. nigricollis (Fig. 3E) venom. To test if heparinoids can block venom cytotoxicity therapeutically, the inventors first treated cells with TV. nigricollis venom and then added heparin over time. Addition of heparin up to 60 minutes after venom could still significantly block venom cytotoxicity (Fig. 3F).[000141] 2,2,3 Heparinoids prevent venom interaction with cell surface[000142] Since heparan sulfate and related molecules bind soluble effectors including growth factors and proteases, the inventors hypothesized that in the context of its venom antidote activity, heparin may act as a “decoy” venom receptor and block venom / host cell interactions. To test this hypothesis, the inventors labelled each cobra venom with an Alexa-488 fluorophore and then evaluated venom / cell interactions by flow cytometry. While labelled cobra venom showed a strong interaction with untreated cells (TV. pallida venom shown in Fig. 4A-C), adding heparin (Fig. 4B) or tinzaparin (Fig. 4c) blocked venom / host cell interactions. These data are quantified in Fig. 4D, and similar results were observed for venom from the two geographical variants of TV. nigricollis (fig. 8A-H). Thus, by flooding the system with free heparin, the inventors can suppress venom / target interactions, and this is sufficient to block cytotoxicity.[000143] 2,2,4 Heparin interacts with three-finger cytotoxins to block venom / host interactions[000144] Snake venoms are variable mixtures of different toxins, and cobra venoms consist predominantly of multiple isoforms of phospholipases A2 (PLA2) and three-finger toxins (3FTx). To identify which specific venom components are sensitive to heparinoids, the inventors separated N. pallida (Fig. 4E and F) and N. nigricollis (fig. 9A and B, and fig. 10A) venoms using fast protein liquid chromatography (FPLC). First, the inventors used heparin affinity chromatography to isolate venom proteins that interact with heparin. Interestingly, most of the venom material bound to the column and was eluted in 3-4 main peaks (Fig. 4E, fig. 9A and fig. 10A), suggesting heparin has a broad capacity to interact with multiple venom components. The main proteins comprising each peak were identified by liquid chromatography mass spectrometry (LC-MS): Pl (weak heparin interaction) contained mainly acidic PLA2, P2 (moderate heparin interaction) contained the 3FTx cytotoxin 1 (CTxl), and P3 (strong heparin interaction) contained both basic PLA2 (bPLA2) and the 3FTx cytotoxins CTx3 and 4. The inventors then further fractionated P3 using cation exchange to separate basic PLA2 (bPLA2) from the 3FTx cytotoxins CTx3 and 4 (Fig. 4F and fig. 9B) and assessed the purity of each fraction on SDS-PAGE (Fig. 4G, fig. 9C, and fig 10B).[000145] Isolated N pallida toxins were then subjected to surface plasmon resonance (SPR) to assess binding affinity with heparin, dalteparin and tinzaparin. Fig. 4H-I shows that tinzaparin binds with sub-micromolar affinity to the fraction containing CTx3 and 4, binds weakly to bPLA2 and exhibits no specific binding to CTxl or PLA2. The same pattern of binding is observed for heparin and dalteparin (fig. 11). Functionally, the 3FTxs CTx3 and 4 were highly cytotoxic and, in line with their binding profile, their activity was significantly inhibited by tinzaparin (Fig. 4J). Similar binding, cytotoxicity and inhibition data were obtained for the two N. nigricollis venoms (fig. 9D-F and fig. 10C-E), with CTx3 and 4 demonstrating the most potent heparinoid binding properties. Collectively, these data demonstrate that heparin and related compounds are acting directly on the cytotoxic 3FTxs CTx3 and 4 from a range of African spitting cobra venoms to block cellular cytotoxicity.[000146] To assess the breadth of this anti-venom activity, the inventors tested the ability of heparin to block other cytotoxic snake venoms (Fig. 4K-O). The inventors found heparin could suppress cytotoxicity caused by venom from the monocled cobra (Fig. 4K; Naja kaouthia), the Chinese cobra (Fig. 4L; Naja atra), and the Indian spectacled cobra (Fig. 4M; Naja naja). However, heparin had no ability to block cytotoxicity caused by West African saw-scaled viper (Fig. 4N; Echis ocellatus) or African puff adder (Fig. 40; Bids arietans) venom. Importantly,while cobra venoms contain cytotoxic 3FTxs, the venoms of these two vipers do not. Overall, these data show that heparin and LMW heparinoid drugs can inhibit cytotoxic 3FTxs and may constitute a new and potent antidote for morbidity-causing cobra venoms.[000147] 2,2,5 Heparinoids protect against spitting cobra-venom induced skin damage[000148] The WHO lists many spitting cobras as “Category 1” species of highest medical importance, because their snakebite leads to severe morbidity-causing pathology, such as local dermonecrosis, and permanent disability. The inventors therefore tested if heparin or heparinoids could protect human epidermal keratinocytes from N. pallida and N. nigricollis (Nigerian and Tanzanian) venom-induced cytotoxicity. Venom from each snake species induced cell death in a concentration-dependent manner (Fig. 5A), and treatment with heparinoids promoted cell survival (Fig. 5B) and inhibited this cell death (Fig. 5C). The inventors next tested the ability of heparinoids to block venom-induced dermonecrosis in vivo using a WHO-recommended preclinical model of local envenoming. Mice were intradermally (ID) dosed with venom from N. pallida, Nigerian N. nigricollis, or Tanzanian N. nigricollis (25, 57, and 63 pg, respectively), preincubated with saline vehicle or the heparinoids dalteparin or tinzaparin (60 pg [3 mg / mL]) (Fig. 5D). While animals injected with venom-plus-vehicle exhibited large dermonecrotic lesions, animals that received venom-plus-dalteparin or -tinzaparin showed significant reductions in lesion sizes, irrespective of the venom or drug tested (Fig. 5E, fig. 12, quantified in Fig. 5F- H). Tinzaparin outperformed dalteparin by providing the greatest reduction in dermonecrosis across the three venoms (mean lesion size reduction of 94% versus 63%, respectively). For these reasons, the inventors progressed tinzaparin into more challenging rescue studies that better reflect envenoming by delivering treatment after venom dosing (Fig. 51).[000149] The inventors used Tanzanian N. nigricollis venom for rescue studies as it was the most dermonecrotic of the three venoms tested (Fig. 5H) and the inventors evaluated the efficacy of ID tinzaparin delivered immediately after venom injection. Both a low dose (3 mg / kg) and moderate ‘human-equivalent’ dose (21.5 mg / kg) of tinzaparin significantly reduced the resulting mean sizes of venom-induced dermonecrotic lesions by 66 and 60%, respectively (Fig. 5J, fig. 13, quantified in 5K). Tinzaparin is an FDA-approved anti -thrombotic that is self-administered subcutaneously (SC) daily. Accordingly, the inventors next challenged mice with the same ID venom dose before immediately delivering tinzaparin SC to a site underneath where venom was injected. The low (3 mg / kg) tinzaparin dose reduced the mean size of resulting dermonecroticlesions by 32% though this trend did not meet statistical significance. However, the moderate (21.5 mg / kg) dose of SC tinzaparin significantly reduced the size of venom-induced dermonecrotic lesions by 50% (Fig. 5L, fig. 13, quantified in 5M) demonstrating that the approved route of administration for this licensed drug is effective at reducing the severity of local envenoming caused by cobra snakebites in vivo. This inhibitory effect was further evidenced histopathologically, as skin tissue samples collected from mice injected with Tanzanian N. nigricollis venom showed prominent damage to all layers, with ulceration of the epidermis and necrosis of the underlying dermis, hypodermis and panniculus carnosus, while mice injected with venom and tinzaparin either preincubated or SC showed substantial reduction in epidermal ulceration and underlying necrosis (Fig. 5N). Overall, these data show that heparinoid drugs are an effective new class of snake venom treatment that act to prevent severe local spitting cobra envenoming by blocking the cytotoxic actions of the 3FTx cytotoxins CTx3 and 4.[000150] 2,3, Discussion[000151] Defining the essential molecular interactions between cytotoxic venoms and target cells provides a fundamental understanding of how these venoms act, and importantly how they can be treated medically. Here the inventors describe multiple critical genes and pathways required for cobra venom cytotoxicity, and for one pathway, heparan / heparin biosynthesis, the inventors characterize this interaction in detail. The unbiased molecular characterization of cobra venom mechanisms led to the surprising discovery of heparinoids as a new, pre-clinically effective, and broad acting class of cobra envenoming antidote. This is the first identification of a much needed cytotoxic 3FTx therapeutic. Overall, the results of this study provide a comprehensive assessment of how to protect from cobra venom, information that will be critical in reducing morbidity in snake envenomings.[000152] The inventors’ findings have direct translational relevance for tropical snakebite envenoming. 3FTxs are highly abundant components of elapid venoms characterized by three loops that connect to a central core. These low molecular mass proteins (~6-9 kDa) have diverse neurotoxic, cardiotoxic and cytotoxic effects. In the context of spitting cobras, 3FTxs are highly cytotoxic by disrupting cell membranes, and cause local tissue necrosis in snakebite victims. Although antibody -based antivenoms are lifesaving therapies, they are ineffective at preventing severe local envenoming. This is likely because of the rapid onset of snake venom-mediatedcytotoxicity, delays in reaching a clinical environment, and the difficulty for centrally delivered antibodies to rapidly penetrate peripherally injured tissue. Thus, in the context of spitting cobra envenoming, there is a strong need for the development of anti-3FTx therapeutics that can be rapidly administered soon after a snakebite, and until now no such inhibitors had been identified. The inventors’ study demonstrates that the LMW heparinoid, tinzaparin, represents an exciting new venom antidote that prevents cytotoxicity and in vivo dermonecrosis caused by cobra venoms from diverse regions of Africa and Asia. Additionally, tinzaparin shows strong translational promise because it is already an FDA-approved therapeutic, which should lead to reduced costs and time associated with its downstream clinical development for snakebite indication. Moreover, it is effective in murine models of envenoming via its approved route of administration; subcutaneous delivery of drug after venom challenge resulted in a significant reduction in venom induced dermonecrosis. The potential for rapid community-level administration of tinzaparin holds much promise for preventing morbidity caused by cobra snakebites.[000153] Overall, the inventors’ findings align with studies investigating the Chinese cobra (N. atra) 3FTx cardiotoxin, as well as several studies using compositionally distinct viper venoms that suggest a protective effect by heparin. For example, preincubation with heparin reduced local skin lesions caused by Russell’s viper (Daboia russelii) venom, and also blocked jararacussu (Bothrops jararacussu) PLA2 myotoxin II damage to muscle or endothelial cells. The inventors’ work reaffirms the protective action of heparin and related compounds, extending it to cytotoxic 3FTxs and provides a molecular mechanism for this activity. Critically, the inventors find the low molecular weight heparinoid, tinzaparin, is suitable to prevent dermonecrosis clinically in a post-envenoming context. Beyond venoms, cellular heparan sulfate has also recently been found to be essential for SARS-CoV-2 infection. Here as well, flooding the system with free heparin or related compounds was sufficient to block infection, and similar observations have been made with other viral and bacterial pathogens. Heparan sulfate proteoglycans are conserved and widely expressed on the cell surface. Thus, targeting these molecules may be an optimal evolutionary strategy to interact with a broad range of species. Conversely, by providing this structure in excess, free heparin / heparinoids may act as a decoy target for multiple unrelated environmental hazards. Overall, the emerging molecular evidence suggests that heparan sulfate is a common cellular entry point for diverse human threats, and heparinoids may have broad untapped activity to protect us.[000154] To date, much of modem molecular medicine has focused on health challenges facing high income countries; however, here the inventors apply these same approaches to understand snakebite envenoming, a significant neglected tropical disease. From these efforts, the inventors identify multiple new genes and pathways essential for snake venom cytotoxicity. For one of these (heparan / heparin sulfate biosynthesis), the inventors exploit this knowledge to generate an affordable, safe, and effective new class of cobra venom antidote that can help in protecting some of the world’s most vulnerable populations.[000155] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall within the spirit and scope of the present invention.

Claims

CLAIMS1. A method of treating or preventing venom induced injury including local tissue damage and / or skin irritation which comprises administering to a subject a composition comprising an effective amount of heparin and / or heparinoid.

2. Use of a composition comprising an effective amount of heparin and / or heparinoid in the manufacture of a medicament for treating or preventing venom induced injury including local tissue damage and / or skin irritation.

3. A composition comprising an effective amount of heparin and / or heparinoid for use in treating or preventing venom induced injury including local tissue damage and / or skin irritation.

4. The method according to claim 1, the use according to claim 2, or the composition according to claim 3, wherein the venom comprises three-finger toxins (3FTxs) and / or one or more other toxin.

5. The method, the use, or the composition according to claim 4, wherein the three-finger toxins (3FTxs) are three finger toxin cytotoxins (or cardiotoxins).

6. The method, the use, or the composition according to claim 4, wherein the other toxins are toxins that interact with or stick to heparin and / or heparinoids.

7. The method according to any one of claims 1 or 4 to 6, the use according to any one of claims 2 or 4 to 6, or the composition according to any one of claims 3 to 6, wherein the heparinoid is a low molecular weight (LMW) heparinoid.

8. The method, the use, or the composition according to claim 7, wherein the LMW heparinoid is a tinzaparin, dalteparin, enoxaparin, or a pharmaceutically acceptable salt thereof.

9. The method according to any one of claims 1 or 4 to 8, the use according to any one of claims 2 or 4 to 8, or the composition according to any one of claims 3 to 8, wherein the composition is administered to a skin of the subject.

10. The method according to any one of claims 1 or 4 to 9, the use according to any one of claims 2 or 4 to 9, or the composition according to any one of claims 3 to 9, wherein the composition is administered topically, subcutaneously, or transcutaneously.

11. The method according to any one of claims 1 or 4 to 10, the use according to any one of claims 2 or 4 to 10, or the composition according to any one of claims 3 to 10, wherein the composition is administered in less than 5 minutes, less than 10 minutes, less than 30 minutes, less than 60 minutes, or about 60 minutes after exposure to the venom.

12. The method according to any one of claims 1 or 4 to 11, the use according to any one of claims 2 or 4 to 11, or the composition according to any one of claims 3 to 11, wherein the composition is a topical formulation, a topical cream formulation, a spray formulation, an injectable formulation, a sunscreen formulation.

13. The method according to any one of claims 1 or 4 to 12, the use according to any one of claims 2 or 4 to 12, or the composition according to any one of claims 3 to 12, wherein the venom induced local tissue damage and / or skin irritation is dermonecrosis.

14. The method according to any one of claims 1 or 4 to 13, the use according to any one of claims 2 or 4 to 13, or the composition according to any one of claims 3 to 13, wherein the venom originates from a siphonophore, jellyfish, an insect, a plant, or a snake.

15. The method, the use, or the composition according to claim 14, wherein the siponophore is Physalia utriculus (blue bottle) or Physalia spp.

16. The method, the use, or the composition according to claim 14, wherein the jellyfish is Chrysaora quinquecirrha (sea nettle).

17. The method, the use, or the composition according to claim 14, wherein the insect is Pristhesancus plagipennis (assassin bug).

18. The method, the use, or the composition according to claim 14, wherein the plant is Urtica ferox (New Zealand stinging nettle).

19. The method, the use, or the composition according to claim 14, wherein the snake is a cobra selected from the group consisting of Nigerian / / a nigricollis (black-necked spitting cobra), Tanzanian Naja nigricollis (black-necked spitting cobra), Naja pallida (red-necked spitting cobra), Naja kaouthia (monocled cobra), Naja atra (Chinese cobra), and Naja naja (Indian spectacled cobra).

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