Our project has several components. The idea was to create a potentially economically viable production platform in Nicotiana benthamiana, testing various Arabidopsis thaliana promoters that respond to different stimuli. The production platform was to be ideally used to produce high levels of therapeutic products, with the appropriate post-translational modifications (PTMs) that makes using plants as a production system more favourable than bacteria. The therapeutic product that we tried to produce was an antagonist to thyroid stimulating hormone, with the aim that this would be able to treat hyperthyroid disorders such as Graves’ disease.
The Therapeutics Part
Hyperthyroidism is the name given to a group of conditions that result in an overactive thyroid gland. Both sexes can suffer from the disease but hyperthyroidism is, according to the NHS, about 10 times more common in women than in men, with the condition usually starting between 20 and 40 years of age. The increased abundance of thyroid hormones can be serious if their levels rise too much, but general symptoms include nervousness, anxiety and irritability, mood swings, fatigue, heat sensitivity, goitre, heart palpitations, trembling, twitching and weight loss. If left untreated, the condition can cause problems with the eyes (bulging and double vision), pregnancy complications such as pre-eclampsia, premature birth or miscarriage, and a potentially life threatening ‘thyroid storm’ when symptoms accumulate rapidly and unexpectedly.
Graves’ disease is an autoimmune disorder that causes the body to attack the thyroid, causing hyperthyroidism as antibodies bind to, and activate, the thyroid gland. The disease is partly heritable, with some associated genes identified, and some environmental factors such as smoking or stress increasing the risk of the disease. The antibody that attacks the thyroid gland is called thyroid stimulating immunoglobulin (TSI), which has a similar effect to thyroid stimulating hormone (TSH). Previous work has shown that a synthetic antagonist (TSHantag) can mitigate the effects of TSI in mice by binding to the thyroid gland, but will not activate it. This prevents the native TSH and TSI binding to the thyroid, and thus should decrease the levels of thyroid hormones in the body, treating the hyperthyroidism.
Our project aims to produce this antagonist in the tobacco leaf expression system and evaluate its potential as a therapeutic agent for Graves' disease. This should be preferable to the current treatment, which is the use of radioactive iodine which destroys the thyroid and renders patients dependent on thyroid medication for the rest of their lives.
The Gene Expression System Part
Our other aim was to generate novel Phytobrick for use in the transient gene expression system in Nicotiana benthamiana. For this, we isolated regions of three plant promoters found in Arabidopsis thaliana . These were constructs with inducible promoters, which were tested using the luciferase expression system. As well as using characterised plant promotors such as 35S or LexA we used these same promotors in an attempt to generate high levels of TSHantag. These constructs can be seen in the diagram below.
The four promoters used were PDF1, PR2, GST6, and WRKY30 (although WRKY30 was later dropped). PDF1 is induced by jasmonic acid, PR2 and GST6 by salicylic acid, and WRKY30 by damage associated molecular patterns (DAMPs) which in this case was the presence of cellulose. We used luciferase to test the promotors' expression levels, and TSHantag variants both with His tags for purification (TSHH). More information about these individual parts can be found on our
basic parts page. More information about our constructs can be found on our composite parts page.
These constructs were created using the Golden Gate Assembly strategy, which involves using type IIS restriction endonucleases to produce sticky ended DNA cuts downstream of a recognition site. As the recognition sequence itself is not cleaved during the process, it allows DNA sequences to be excised and ligated to another DNA sequence with matching sticky ends. Different DNA sequences are synthesised to incorporate these sticky ends, allowing ligation of multiple different DNA fragments in a specific order simultaneously. It can also be set up to allow the restriction site to be eliminated from the ligated product, allowing digestion and ligation to be carried out simultaneously. This is shown diagrammatically.
For our project, the type IIS restriction enzymes that we used were BsmB1 for assembly into a level 0 plasmid (pSB1C3), and Bsa1 for assembly into level 1 plasmids (pGB-A1, pGB-A2). Their recognition sites are as follows, where "|" is the site of endonuclease activity: