CRISPR Identifies Common Blood Thinner as Snake Venom Antidote

Each day, 7,400 people are bitten by a snake, resulting in 81,000–138,000 unfortunate deaths per year, according to the World Health Organization (WHO).

While antibody-based antivenoms exist, they can be difficult to access in low-income countries, and are not always effective against local tissue injury, which contributes heavily to mortality rates. In 2017, the WHO added snakebite envenoming to its highest priority neglected tropical disease (NTD).

“Snakebites remain the deadliest of the NTDs, with its burden landing overwhelmingly on rural communities in low- and middle-income countries,” said Professor Nicholas Casewell, head of the Centre for Snakebite Research and Interventions at Liverpool School of Tropical Medicine.

Using CRISPR gene-editing technology, researchers at the University of Sydney and Liverpool School of Tropical Medicine have demonstrated how a commonly used blood thinner could be repurposed as a cost-effective and easy-to-access cobra venom antidote. The research is published in Science Translational Medicine.

Repurposing existing drugs for snakebites using CRISPR technology

As the health and economic burden of snakebite envenoming grows, researchers are exploring how drug repurposing could provide readily available and cost-effective treatment approaches. A challenge in this arena is building a knowledge base on how venoms interact with the human body at the molecular level to test appropriate drugs.

CRISPR gene-editing technology is extremely useful in this context. CRISPR screens offer a high-throughput method for evaluating the role of specific genes in cellular processes or phenotypes. Equipped with this knowledge, researchers can evaluate the effectiveness of existing or novel drugs for blocking these interactions.

Tian Du, a PhD student from the University of Sydney, and colleagues conducted a whole-genome CRISPR knockout screen in human cell lines after administering venom from different types of cobras. “We used a functional genomics approach to define venom–target genetic interactions that modify the cytotoxicity caused by spitting cobra venom and then used this information to develop a locally acting venom antidote,” the authors said.

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“Our findings are exciting because current antivenoms are largely ineffective against severe local envenoming, which involves painful progressive swelling, blistering and/or tissue necrosis around the bite site,” said Casewell, who is a co-author of the paper. “This can lead to loss of limb function, amputation and lifelong disability.”

Heparinoid reduces tissue death after venom injection in mice

Several genes, including EXT1, B4GALT7, EXT2, EXTL3, XYLT2 and NDST1, were highlighted in the CRISPR screens.

These genes encode enzymes that are involved in the production of heparan and heparin; the former is located on the surface of cells and in the extracellular matrix, while the latter is stored in immune cells and released into the bloodstream during an immune response. The similar structure of these molecules means that the snake venom tested was capable of binding to both.

“To validate these results, we generated single knockout cell pools with single guide RNAs that targeted each resistance gene individually and tested cytotoxicity. Targeting each component of the heparan/heparin biosynthesis pathway conferred some resistance to each venom, confirming a role for heparan in cobra venom cytotoxicity,” Du and colleagues said.

As heparan is necessary for spitting cobra venom to cause cytotoxicity, flooding the site of the bite with “decoy” heparin, or heparinoids – low molecular weight variants of heparin – could work therapeutically to block it, the researchers hypothesized.

After encouraging results in human cell lines, they moved on to in vivo experiments testing heparin and heparinoids as venom antidotes in mice. The US Food and Drug Administration (FDA)-approved heparinoid, tinzaparin, reduced the size of dermonecrotic lesions when administered subcutaneously at the same time as an intradermal venom injection.

This drug has the most translational value, the researchers argue, given that it already has received approval for human use in other indications.

“Heparin is inexpensive, ubiquitous and a WHO-listed Essential Medicine. After successful human trials, it could be rolled out relatively quickly to become a cheap, safe and effective drug for treating cobra bites,” Du said.

As tinzaparin did not block dermonecrosis completely, further preclinical studies evaluating dosing, delivery route and potential toxin-targeting drug combinations are warranted.

Time is of the essence here, Professor Greg Neely, a corresponding author of the study from the Charles Perkins Centre and Faculty of Science at the University of Sydney, emphasized, considering the WHO’s 2030 goal: “That target is just five years away now. We hope that the new cobra antidote we found can assist in the global fight to reduce death and injury from snakebite in some of the world’s poorest communities.”

The research team believe CRISPR screens will prove useful in future studies exploring the molecular mechanisms of envenoming. While this study homed in on the genes involved in heparan and heparin biosynthesis, others were identified in the initial screening process that could be implicated in cytotoxicity.

“Cytotoxicity is only one physiologically relevant impact of snake envenoming, and further CRISPR screening using other functional readouts beyond cell death may provide a more comprehensive understanding of mechanisms of action underlying envenoming,” the authors concluded.

Reference: Du TY, Hall SR, Chung F, et al. Molecular dissection of cobra venom highlights heparinoids as an antidote for spitting cobra envenoming. Sci Trans Med. 16(756):eadk4802. doi: 10.1126/scitranslmed.adk4802