Determining the embryonic mechanisms and routes to prevention of a birth defect caused by pregnancy

Determining the embryonic mechanisms and routes to prevention of a birth defect caused by pregnancy exposure to an anti-retroviral drug for maternal HIV infection

Background:
Women who are HIV-positive receive antiretroviral therapy (ART) in pregnancy to prevent vertical HIV transmission to the baby. ART carries the risk of potentially damaging the developing fetus.
Dolutegravir (DTG), an HIV integrase strand transfer inhibitor, is an effective ART drug, and is used in pregnancy [1]. However, a study of DTG usage in Botswana revealed a higher than expected rate of neural tube defects (NTDs) among pregnancies exposed to DTG from conception [2]. Other birth defects also show an increased rate: e.g. the anterior body wall (ABW) closure defect, omphalocele
(https://www.cdc.gov/ncbddd/birthdefects/omphalocele.html) is commoner in women taking DTG from conception [4]. Further research is needed to determine how DTG may harm the developing fetus.

Aims/Objectives:
This project aims to determine the developmental mechanisms by which DTG causes defects of ABW closure in mouse fetuses. The mice will also have dietary folate deficiency, as we have found this to increase ABW defect frequency significantly. The student will carry out research into:

• The developmental time-course of ABW defects in mouse fetuses
• Similarities and differences between DTG-induced and genetically-caused ABW defects
• Potential interventions in pregnancy that can prevent defects of ABW development.

Methods:
This PhD project is based in our lab at ICH, where we have long experience of genetic and developmental biology studies of birth defects [3]. The student will learn methods of mouse breeding, genetics and fetal dissection, and will collect a series of fetal stages to understand the normal process of gut herniation. This occurs at embryonic days 14-16, when the midgut leaves the body cavity, rotates through 270o, and returns to the cavity [4]. ABW defects arise when the gut stays outside the body (omphalocele). The mechanisms by which DTG may cause ABW defects will be studied by determining: (i) Tissue structure as determined by fetal dissection and histology; (ii) Cell proliferation - by phospho-histone H3 immunostaining to reveal mitotic cells, with calculation of cell proliferation rates; (iii) Cell death - by anti-activated caspase 3 immunostaining for cells in apoptosis; (iv) Gene expression analysis – to analyse genes known to be involved in ABW defects in other mouse mutant strains [4]. A comparison will be made with ABW defects in the Scribble mutant mouse [5], to determine if similar mechanisms apply when different causal factors are present. Possible preventive methods will be explored, depending on the main tissue, cellular, or molecular defect that is identified. For example, if apoptosis is found to be increased in amount, as previously [4], then an anti-apoptotic chemical inhibitor will be used to suppress cell death. 

Collaboration with University of Toronto:
The supervisor in Toronto (Dr Lena Serghides) is highly experienced in studies of anti-HIV drugs in pregnancy and has supervised more than 20 research fellows and students. The student will travel to Toronto, after initial training at ICH, to collect fetuses with ABW defects and normal controls, from DTG-treated mice. The Toronto lab has great experience of mouse DTG treatment, at dose levels that produce similar blood concentrations as in women on anti-HIV therapy.

Timeline:
Months 1-6 will involve initial training at ICH. Months 7-12 will be spent in the Toronto lab, to collect a series of staged fetuses after DTG treatment of pregnant females. The student will return to ICH for
months 13-30, when analysis of the fetuses, and new intervention experiments will be done. Months 30-36 are for completing analysis and writing the PhD.  

References:
[1] Hill. J Virus Erad 4, 66-71 (2018). [2] Zash et al. N. Engl. J. Med 381, 827-840 (2019). [3] Copp et al. Nat Rev Dis Primers 1, 15007 (2015). [4] Brewer & Williams. BioEssays 26, 1307-21 (2004).
[5] Murdoch et al. Hum Mol Genet 12, 87-98 (2003).