Antiphospholipid syndrome (APS) is an autoimmune disease. This means that it is a disease in which the immune system of the body, which is designed to protect us against infections, instead starts to attack parts of the body itself causing the disease process. Different autoimmune diseases attack different parts of the body and have different symptoms. In APS, the problem is that the immune system makes antibodies called antiphospholipid antibodies (aPL) which interact with various different types of cells. The main cells affected are in blood vessels or in the womb, so the main effects of APS are to cause clots in blood vessels, strokes in the brain and/or recurrent miscarriages. APS is one of the main causes of these problems; for example it is one of the most important causes of stroke in people under 50. The only treatments currently available to prevent clots, strokes or miscarriages in patients with APS are drugs that thin the blood and stop it from clotting. These drugs are called anticoagulants, and include warfarin and heparin. However, they have side-effects, notably a risk of bleeding, because they oppose all clotting - even the helpful clotting that occurs after an injury to stop bleeding from a wound. We seek to develop an entirely new form of treatment for APS, which does not thin the blood but which directly targets the aPL themselves. The main way in which aPL cause their harmful effects in APS is to attach themselves to a protein in the blood called beta-2-glycoprotein I (beta2GPI). Beta2GPI is present in everyone and is harmless in the absence of aPL. When aPL combine with beta2GPI, however, this combination can bind to the surfaces of cells in the blood vessels or womb, change the behaviour of these cells and thus promote clotting or miscarriage. We are developing a drug that will be designed to stop aPL binding to beta2GPI to prevent this harmful process from occurring. Beta2GPI is composed of five parts, called domains, arranged end to end like beads on a string. We know that aPL primarily attach to the end domain (Domain I or DI). Over the last 10 years our research group has developed the only system in the world for making DI in bacteria. We are now able to grow these bacteria in large quantities and purify DI from the bacterial cultures. This can be done in high-yield with the DI at over 95% purity. We have shown that this purified DI can be used to block binding of aPL from patients with APS to human beta2GPI on plastic plates and also to stop human aPL from causing clots in mice. However, DI is a small molecule, which makes it unsuitable for use as a drug because it would only be retained in the body for a few hours. To circumvent this problem we need to modify our DI to make it larger. We are doing this by a process called PEGylation, in which large polyethylene glycol (PEG) molecules are joined to smaller molecules. We have been working with a biotechnology company called PolyTherics to achieve this. PolyTherics have developed technology to PEGylate small molecules at precisely determined points on their surface. We have achieved production of three different variants of PEGylated DI, which have PEG of different sizes. Larger PEGs could be good to make the DI last longer in the body after injection but could also block the effects of DI on aPL. Therefore we need to do tests comparing all three variants to see which is best. We have already proved that our PEG-DI blocks effects of aPL from patients with APS on binding to beta2GPI, on clotting in a test tube and on formation of clots in mice. In this project we will carry out further tests to find out which form of PEG-DI is best at blocking effects of aPL then take that form forward to tests in animals. These tests will determine how long it is retained in the body and whether it has any toxic side-effects. Assuming no toxicity is found we will develop production of this PEG-DI at large scale in a form pure enough for human trials.

Our experiences in early life can have life-long effects on our health and wellbeing. For example, in a rural population in The Gambia in West Africa we have observed that children born in the rainy season are 6 times more likely to die between the ages of 15 and 65 than those born in the dry season. In fact there is mounting evidence that detrimental influences on lifelong health can stretch right back to the early stages of embryonic development. This underlines the importance of research into the underlying mechanisms, so that the processes linking environmental exposures to negative outcomes can be understood, and hopefully corrected. One such possible mechanism involves a process known as methylation, which is one type of 'epigenetic' modification of the genome. Methylation requires a defined set of nutrients including folic acid and B-vitamins, both to provide the necessary chemical compounds, known as methyl groups, and to undertake the necessary metabolic conversions. Animal experiments have previously shown that supplementing the diets of female mice with these nutrients before they conceive has a profound effect on their offspring's appearance (e.g. changing their coat colour) and that these changes were associated with higher levels of methylation on their DNA. Until now it was unknown whether similar effects on offspring methylation occur in humans, but our group recently presented first-in-human evidence that they do. We have since followed up this work by looking at patterns of methylation across the genome. We found evidence of unusual or 'disrupted' methylation patterns associated with both maternal nutrient status and season of conception in certain types of genes, and notably in one gene (VTRNA2-1), where disrupted methylation has previously been linked with some forms of cancer and also with negative effects on the immune system. With this grant we hope to extend this work in a number of ways. Firstly, we want to characterise these patterns of disruption more precisely by looking at a larger number of infants; interrogating key regions of the epigenome at high resolution using more advanced technologies; and looking for methylation effects all the year round. We hope this will provide further clues about the mechanisms underlying epigenomic disruption. Secondly, we want to investigate the effects of disrupted methylation in VTRNA2-1. Our Gambian research centre is a particularly good place to do this as we are able to link an individual's epigenetic information with medical records and other demographic data, and we can also conduct detailed laboratory investigations on blood cells in individuals known to have abberant methylation. These functional studies are an important part of the chain linking epigenetic effects to real, adverse health outcomes in people. Finally, with the help of advanced computer modelling, we will identify the specific combination of MD-related nutrients that may be causing the observed patterns of disrupted methylation. We will then develop a nutritional supplement to correct the observed suboptimal nutrient profile, and we will test its effectiveness in a randomised controlled trial. If effective, in future work we would seek to assess the effect on offspring methylation of giving this supplement to mothers-to-be. The hope is that the patterns of disrupted methylation previously observed in infants conceived at certain times of the year would then be prevented. In the longer term, we hope that the work described here will inform strategies for pre-conceptional supplementation in mothers that will lead directly to improved outcomes for infant growth and development, with life-long benefits for health and wellbeing.