Production of Neosaxitoxin using Genetic Methods

Natural toxins have often provided major socioeconomic benefits in the medical industry. Saxitoxins, commonly known as paralytic shellfish toxins (PSTs), are a group of neurotoxic alkaloids produced by algal blooms, i.e. marine dinoflagellates (red tide) and freshwater filamentous cyanobacteria that can block neuronal communication for short periods. This property can be exploited in the medical industry for acute situations such as post-operative pain management and itching. Neosaxitoxin is a natural, N-1-hydroxyl variant of saxitoxin that shows higher affinity for sodium channels and is more efficacious. Recent clinical trials have indicated a positive safety profile for neosaxitoxin in humans as a long-acting local anesthetic. The major challenge with this class of compounds is their inadequate availability due to expensive synthetic processes and small yields. Commercial production relies at present on extraction methods from natural producer organisms (cultured microalgae) or from shellfish that have been exposed to toxic algal blooms. Cultivation of toxin-producing microalgae is difficult and costly even at small scales, and yields only small quantities. Thus, the adoption of neosaxitoxin has been affected by the lack of an industrial process of production. The unavailability of such a production process is still unresolved despite the immense market potential. Modern molecular technologies can re-define the process to manufacture clinical-grade neosaxitoxin. This project will express the entire neosaxitoxin biosynthetic pathway genes in two different heterologous systems. Preliminary evidence shows that these genes can be expressed heterologously and display function in bacteria. In summary, the proposed method will provide for a simpler, cleaner and more effective process compared to today's state of the art. We have accomplished our main aim, which was to demonstrate that neosaxitoxin can be produced in bacteria by heterologous gene expression. Neosaxitoxin production was verified by accurate mass LC-MS, as well as by biological (mouse neuroblastoma assay) and immunological (ELISA) assays. During the course of the project, we have filed a patent application (priority date 12.2.2016), which was now submittet as an international (PCT) patent application. We are currently establishing a new company, Neosynthetica AS, which will be owned 51% by the Norwegian side and 49% by our Australian partners of the research consortium. Finally, we have established contact and signed confidentiality agreements with two important stakholders, which are the Boston Childrens Hospital and Grünenthal.

Natural toxins have often provided major socioeconomic benefits in the medical industry. Saxitoxins, commonly known as paralytic shellfish toxins (PSTs), are a group of neurotoxic alkaloids produced by algal blooms, i.e. marine dinoflagellates (red tide) and freshwater filamentous cyanobacteria that can block neuronal communication for short periods. This property can be exploited in the medical industry for acute situations such as post-operative pain management and itching. Neosaxitoxin is a natural, N-1-hydroxyl variant of saxitoxin that shows higher affinity for sodium channels and is more efficacious. Recent clinical trials have indicated a positive safety profile for neosaxitoxin in humans as a long-acting local anesthetic. The major challenge with this class of compounds is their inadequate availability due to expensive synthetic processes and small yields. Commercial production relies at present on extraction methods from natural producer organisms (cultured microalgae) or from shellfish that have been exposed to toxic algal blooms. Cultivation of toxin-producing microalgae is difficult and costly even at small scales, and yields only small quantities. Thus, the adoption of neosaxitoxin has been affected by the lack of an industrial process of production. The unavailability of such a production process is still unresolved despite the immense market potential. Modern molecular technologies can re-define the process to manufacture clinical-grade neosaxitoxin. This project will express the entire neosaxitoxin biosynthetic pathway genes in two different heterologous systems. Preliminary evidence shows that these genes can be expressed heterologously and display function in bacteria. In summary, the proposed method will provide for a simpler, cleaner and more effective process compared to today's state of the art.