“New genome data paves a path for making safe and effective synthetic antivenom”March 7, 2020
Dr Sekar Seshagiri, President, Science & Education SciGenom Research Foundation (SGRF), Bengaluru, led the Indian scientists who assembled the genome and transcriptome sequence of the Indian Cobra. Edited excerpts from an interview:
The Indian cobra genome project represents a significant scientific advance in creating a high-quality reptilian reference genome. What are its implications?
Four bases, G A T and C form the chemical alphabet that make up the DNA sequence and serves as the blueprint of a given organism. DNA is present inside nearly all cells in a living organism and is partitioned and kept in chromosomes. Together, the chromosomes represent the genome. The Indian cobra has 19 pairs of chromosomes, 18 autosomes and 2 pairs of sex chromosomes. Ultimately, the goal of any genome sequencing project is to figure out the sequence of chromosomes from end to end. With the aid of cutting-edge genomics technologies and several computational tools, we are able to generate a near-chromosome sequence for the Indian cobra. Starting from DNA molecules of varying sizes that were extracted from tissues, we were able to assemble 19 very large pieces (‘scaffolds’) that roughly corresponds to the chromosomes in this species. These large scaffolds allowed us to then search the genome for the code for protein coding genes. This process is referred to as genome annotation. Next, we asked which of these are expressed in a given tissue, i.e., made into RNA that then is used to produce proteins. Using the comprehensive genome annotation, the expression patterns of different genes across tissues and similarity to toxins, we have identified 19 relevant toxins in the Indian cobra genome that are highly expressed in the venom gland of this animal. This knowledge now allows us to produce these proteins using recombinant protein production techniques, much like how the human insulin protein is made in bacteria. Once we make the proteins, we can then use a technique called the ‘phage display’, that won the 2018 Nobel prize in Chemistry, to develop antibodies that can bind to toxins. A cocktail of such antibodies for the most important/toxic venom proteins should be able to neutralize the often-lethal effects of snakebite in victims.
This would be a significant advance in healthcare, given that the current standard of care for snakebite involves a process that is over 100 years old. Currently, large mammals like horses are injected with venom extracted from snakes (a process called ‘milking’) and the antibodies produced are then purified from the blood and sold as antivenom. However, over 70% of the antibodies from the horse has nothing to do with the injected snake venom. Many of these antibodies are made in response to the various environmental stimuli a horse may be typically exposed to (bacteria etc.). Thus, when patients are given antivenom, they are getting a lot of non-specific horse antibodies. These horse antibodies are known to cause ‘serum sickness’ that can lead to kidney failure and severe allergic reactions. The genome and accompanying information generated in this study paves a path for making synthetic antivenom that will be safe and effective.
In addition to this, the venom itself is a source of potential drugs like those used to lower blood pressure or treat blood clotting issues that lead to heart attacks. Having a complete catalogue of the components of the venom will provide templates for drugs. Also, the techniques developed for generating the high-quality Indian cobra genome can be extended to other animals.
How do you decide what are the relevant toxins in the venom?
Gene prediction tools are commonly used to identify protein coding genes in a genome. Proteins are often conserved between closely related species. This helps us ascribe a certain function to a gene. Some maybe involved in skin development, some in metabolism, and so on. Using algorithms, we identified genes that most resembled toxins identified in other venomous species. Armed with this information, we can then begin to understand the role of these genes, where they are expressed among other things. Genes are expressed at different levels in different tissues throughout the body. For example, insulin is only expressed in one cell type in the body, that is the beta cells in the pancreas. In the case of the Indian cobra, using gene expression data from 14 different tissues, we were able to identify genes that were expressed in some tissues only while not detected in other tissues. Of particular interest to us, we wanted to know which genes were highly expressed in the venom gland – where venom is produced. Using statistical methods, we were able to narrow down a set of 19 genes that were expressed uniquely in venom gland. These 19 genes encoded for proteins that were highly similar to known venom proteins catalogued in other snakes. These toxins had a range of different physiological effects (neurotoxins, cytotoxins, etc.).
How challenging was the sequencing project?
The challenge with a sequencing project such as this is the fact that there is no prior knowledge about the genome. Typically, genome sequencing projects benefit from knowing answers to questions such as: how many chromosomes are there in the genome of interest? How big is the genome (E.g., the human genome contains ~3 billion nucleotide base pairs)? How many genes are present in this genome? Once we have the genome and genes, the next important task is finding out which genes are expressed in different tissues. In this case, we were most interested in identifying the minimum set of venom gland-specific genes. Prior to this study, not a lot was known from a snake genomics perspective. Only a handful of low-to-middling quality snake genomes were publicly available. So, we implemented a comprehensive computational pipeline to not only create a high-quality genome, but also to identify the venom genes in this species. We have now identified genes that are likely key contributors to venom-induced effects in snakebite victims. This will help us generate antibodies using phage display technology as mentioned above. We anticipate that it may take about 2-3 years to translate this research into producing a safe and effective antivenom.
Will you partner with anyone to support the antivenom project?
There are a few antivenom companies in India which are interested, however, there is also a lot of interest from academia. Several researchers across India are working in this area. Recently, the Wellcome Trust announced funding to support antivenom development encouraging academia-industry collaborations to solve this neglected tropical disease. One of the biggest reasons why the field of antivenom is lagging behind the pharma industry is because antivenom is not a biotherapeutic. Compared to finding a cure for a disease such as cancer, snakebite is a problem in underdeveloped and developing nations. Therefore, pharma companies are not particularly interested in it because antivenom does not generate sufficient revenue. A well-known example is when Sanofi, citing a lack of a lucrative market, stopped manufacturing and supplying Fav-afrique, a highly successful antivenom against several deadly African snakes. We feel that given the enormity of the problem faced by developing nations, and recent upsurge in interest from academia, non-profit organizations as well as the World Health Organization, we are happy to establish consortia to set out a roadmap to combat snakebites.
With inputs from Kushal Suryamohan, Bioinformatics Scientist, MedGenome Inc., USA, one of the authors of the paper published in Nature Genetics.