title: Investigating placental MicroRNAs in preeclampsia and optimising a gene therapy strategy for targeting the placenta
Creators: name: Frazier, Sonya Elena
Publisher: name: University of Glasgow
Year: 2021
Subjects: subject: placenta, microRNA, placental microRNAs, preeclampsia, ultrasound, microbubbles, gene therapy, ultrasound-mediated gene delivery.

description: Improvements in the diagnosis and management of preeclampsia (PE) have contributed to a reduction in maternal mortality in parts of the world. However, current treatments still carry safety and efficacy issues, and perinatal morbidity and mortality remains prevalent. Research into the pathology of PE and new treatment strategies are therefore warranted. Gene therapy represents a potential treatment strategy since PE is now recognised as a complex genetic disorder. Despite the clinical progress of gene therapy in foetal growth restriction, less invasive and non-viral delivery systems targeting the placenta are required to mitigate safety concerns and extend research to other pregnancy disorders, such as PE. Identifying a therapeutic gene target in PE remains a challenge since a lack of replication in gene association studies suggests rare variants with small effect sizes likely contribute to the development of PE. Alternatively, a growing body of evidence suggests placental microRNAs (miRNAs) play a key role in the pathology of PE and may therefore represent therapeutic targets. Preclinical studies however remain limited, and inconsistencies between clinical studies make it difficult to discern placental miRNA profiles characteristic of PE. Therefore, in addition to investigating non-invasive and non-viral delivery systems for gene therapy purposes in PE, there is a need for further preclinical and clinical studies to examine the role of placental miRNAs in PE and evaluate their therapeutic potential. Ultrasound-mediated gene delivery (UMGD) can be utilised for non-viral and non-invasive gene transfer, a method in which ultrasound (US) stimulates vector uptake through its mechanical effects following interaction with US contrast agents known as microbubbles. Although the technique has been investigated in a variety of tissues, research into targeting the placenta is limited, with a single UMGD study in baboons published to date. Chapter 3 sought to develop and optimise an in vivo UMGD protocol in rodents that stimulates targeted transfer of a reporter gene to a surrogate organ which could then be applied to the placenta, given the difficulty in targeting a specific placenta in litter-bearing species. These optimisation studies allowed issues to be identified and logical modifications to be made to the protocol, including the type and size of vector employed, the plasmid DNA and MB dose, and the organ targeted. Through these studies, a protocol was established showing evidence of gene transfer of a luciferase plasmid to mouse hearts, allowing progression to a proof-of-concept study. Chapter 4 sought to demonstrate proof-of-concept that UMGD can achieve tissue-specific transfer of an expression vector and is therefore suitable for targeting placenta. UMGD of a luciferase plasmid to mouse hearts showed luciferase activity was significantly greater in the ventricles compared to non-target organs (liver, lungs, and spleen) (p&lt;0.05), providing evidence of tissue-specific transfer. Although there was a trend towards greater luciferase activity in the ventricles compared with the remaining non-target organs (skeletal muscle, left kidney, and right kidney), this was non-significant. Due to small n-numbers, significance testing was not performed comparing tissue from the negative control mice with UMGD treatment mice. The protocol was subsequently applied to pregnant mice to target the placenta. Low levels of luciferase gene transfer were evident in the placenta positioned closest to the cervix on the targeted left uterine horn. Non-target tissues were not evaluated for gene transfer due to time constraints, meaning this aspect of safety could not be confirmed. Placental-specific delivery of an expression vector with UMGD therefore remains to be demonstrated in order to support the clinical translatability of this technique. Identifying a placental miRNA dysregulated in preeclamptic patients or a subset of patients remains essential to identifying miRNAs that represent a clinically relevant therapeutic target. Animal models on the other hand provide a means of exploring the role and therapeutic potential of miRNAs in PE. In Chapter 5, a literature review was conducted to collate evidence from patient studies and identify clinically relevant miRNAs of interest for therapeutic targeting. Subsequently, the expression of candidate miRNAs was evaluated in whole placental tissue and individual placental layers of a rat model of superimposed PE (SPE) previously established by our group. The literature review identified placental miRNAs consistently detected as significantly differentially expressed by miRNA expression profiling studies in third trimester preeclamptic patients. Clinical studies were either in agreement or discordant in the direction of expression of the miRNAs in PE. Evaluation of candidate miRNAs in the placenta of the SPE rat model contributed to the growing body of evidence that placental miRNAs differ between PE subtypes and placental layers, also providing novel evidence of dysregulation of miRNAs within placental layers in rats, a finding previously only shown in humans. Furthermore, four miRNAs (miR-210-3p, miR-223-3p, miR-181a-5p, and miR-363-3p) were identified as potential therapeutic targets given their consistent dysregulation in PE. MiRNA expression profiling allows identification of differentially expressed placental miRNAs in a hypothesis free manner. In Chapter 6, miRNA sequencing was performed to identify differentially expressed placental miRNAs in the rat model of SPE, representing the first study to conduct miRNA sequencing in the placenta of an animal model of PE. Furthermore, the dysregulation of select miRNAs and predicted gene targets was examined by RT-qPCR in placental tissue from the SPE rat model as well as preeclamptic patients to evaluate the clinical relevance of the findings. Expression of miR-210-3p and its predicted gene target, fibroblast growth factor receptor-like 1 (FGFRL1), demonstrated an inverse relationship in the placentas of the SPE rat model and preeclamptic patients as well as in a BeWo trophoblast cell line. This is in agreement with published data that show miR-210-3p is upregulated in PE and provides novel evidence of altered FGFRL1 expression in the placentas of preeclamptic patients. This adds to the existing literature that suggests miR-210-3p plays a pathological role in PE and is a promising therapeutic target. This thesis has provided information on a non-viral and non-invasive gene therapy technique for targeting the placenta as well as miRNAs dysregulated in the placenta in PE and potential therapeutic targets. The development of safe and efficient delivery systems that target the placenta and the identification of suitable targets represent key aspects to establishing placental gene therapy as a treatment strategy in PE. The work in this thesis has provided means to further investigate UMGD for targeting the placenta in pregnant mice, important preclinical models in pregnancy research. Furthermore, the work in this thesis has identified aspects of placental miRNAs that should be considered in future work seeking to investigate the role of and/or target placental miRNAs. Finally, the work in this thesis has identified several miRNAs that are potentially therapeutic targets in PE.