Investigating placental microRNAs in preeclampsia and optimising a gene therapy strategy for targeting the placenta

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<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

From animal models to patients : the role of placental microRNAs, miR-210, miR-126, and miR-148a/152 in preeclampsia

Placental microRNAs (miRNAs) regulate the placental transcriptome and play a pathological role in preeclampsia (PE), a hypertensive disorder of pregnancy. Three PE rodent model studies explored the role of placental miRNAs, miR-210, miR-126, and miR-148/152 respectively, by examining expression of the miRNAs, their inducers, and potential gene targets. This review evaluates the role of miR-210, miR-126, and miR-148/152 in PE by comparing findings from the three rodent model studies with in vitro studies, other animal models, and preeclamptic patients to provide comprehensive insight into genetic components and pathological processes in the placenta contributing to PE. The majority of studies demonstrate miR-210 is upregulated in PE in part driven by HIF-1a and NF-?Bp50, stimulated by hypoxia and/or immune-mediated processes. Elevated miR-210 may contribute to PE via inhibiting anti-inflammatory Th2-cytokines. Studies report an up- and downregulation of miR-126, arguably reflecting differences in expression between cell types and its multifunctional capacity.MiR-126 may play a pro-angiogenic role bymediating the PI3K-Akt pathway. Most studies report miR-148/152 family members are upregulated in PE. Evidence suggests they may inhibit DNA methylation of genes involved in metabolic and inflammatory pathways. Given the genetic heterogeneity of PE, it is unlikely that a single placental miRNA is a suitable therapeutic target for all patients. Investigating miRNAs in PE subtypes in patients and animal models may represent a more appropriate approach going forward. Developing methods for targeting placental miRNAs and specific placental cell types remains crucial for research seeking to target placental miRNAs as a novel treatment for PE

Ultrasound and microbubble gene delivery for targeting altered placental microRNAs in preeclampsia

Ultrasound (US) and microbubble (MB) gene delivery has attracted growing interest as a clinically applicable gene therapy (GT). Though preclinical studies have investigated the system in various tissues, there is limited research in targeting the placenta. This is a potential therapeutic strategy for preeclampsia (PE), which has an underlying genetic basis and ineffective management strategies. Differentially expressed placental microRNAs (miRNAs) in PE may represent suitable targets for GT. Microbubbles (SonoVue) and plasmid (pGL3 or pGL4.13) were administered systemically to CD1 mice, followed by exposure of the heart to US (H14, 1.8 M.I., 1cm focal depth, 2 minutes), using Siemens Acuson Sequoia-512 system and 15L8 probe. Luciferase assays were performed to evaluate gene transfection. Significantly differentially expressed placental miRNAs in PE patients were identified as candidates based on detection by three or more screening studies. Expression of candidate miRNAs was measured by qRT-PCR in PE rat model placentas. In trial 1, low levels of luciferase activity were detected in the heart of treatment mouse 1, 2 and 3. In trial 2, luciferase activity was evident in the atria of treatment mouse 2. In trial 3, higher luciferase activity was detected in the ventricles of the treatment mouse and activity was also detected in the atria. The literature review identified eight candidate miRNAs. MiR-223 (1.46-fold increase) and miR-181a (0.81-fold decrease) were significantly differentially expressed in PE rat model placentas. MiR-223 and -181a may represent targets for US and MB gene delivery. Future studies will apply the US and MB gene delivery protocol for translation to targeting the placenta in our PE rodent model

To be understood: Transitioning to adult life for people with Autism Spectrum Disorder

Introduction: The purpose of this study was to explore the viewpoints of parents of young people with Autism Spectrum Disorder (ASD) in relation to their child's transition to adulthood. Methods: Data were collected during four structured focus groups with 19 parents of young people with ASD with average to high intellectual capacities. Condensed meaning units were identified and checked during focus groups, and were subsequently linked to the International Classification of Functioning, Disability and Health (ICF). Results: Three major themes emerged: to be understood, to understand the world and to succeed. The ICF domains of activity and participation and environmental factors emerged as having the greatest potential to influence transition outcomes. Conclusions: Policies and services should focus on strengths to maximise participation in higher education, employment and independent living amongst young people with ASD. Interventions targeting environmental factors could be effective in improving participation in adult life. Person-centred and individualised approaches could further complement this approach supporting the transition to adulthood for people with ASD, ultimately improving outcomes in adulthood

Students as co-creators of an inclusive equality and diversity teaching resource: an example from life sciences

No abstract available

UofG Equality and Diversity in the Life Sciences: University of Glasgow app

The Equality and Diversity (E+D) in the Life Sciences app has been designed as a learning and teaching resource to raise awareness of issues in academic environments and beyond. This course was designed to make our Life Sciences curriculum at the University of Glasgow more inclusive and empowering for students, allowing them to break down any potential barriers they may come across in their career.  The resource has been built around the 9 protected characteristics embedded in the Equality Act, 2010, namely Age, Disability, Gender Reassignment, Marriage and Civil Partnership, Pregnancy and Maternity, Race, Religion and Belief, Sex. The main aim is to equip students with the necessary knowledge about E+D related issues by supporting the development of critical consciousness and thinking in students, by sharing good practice and encouraging a mindset change about E+D.   This app includes 10 problem-based learning case studies, focusing on the different protected characteristics in the Equality Act, 2010, and varying Life Science career paths. The case studies (termed Bio) are linked to Intended learning Outcomes (ILOs), with complementary tasks and resources, allowing students to explore different topics of interest. There are role model interviews that compliments these case studies and are inspirational in their nature

Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection

INTRODUCTION: Immunological memory is the basis for durable protective immunity after infections or vaccinations. Duration of immunological memory after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and COVID-19 is unclear. Immunological memory can consist of memory B cells, antibodies, memory CD4+ T cells, and/or memory CD8+ T cells. Knowledge of the kinetics and interrelationships among those four types of memory in humans is limited. Understanding immune memory to SARS-CoV-2 has implications for understanding protective immunity against COVID-19 and assessing the likely future course of the COVID-19 pandemic. RATIONALE: Assessing virus-specific immune memory over at least a 6-month period is likely necessary to ascertain the durability of immune memory to SARS-CoV-2. Given the evidence that antibodies, CD4+ T cells, and CD8+ T cells can all participate in protective immunity to SARS-CoV-2, we measured antigen-specific antibodies, memory B cells, CD4+ T cells, and CD8+ T cells in the blood from subjects who recovered from COVID-19, up to 8 months after infection. RESULTS: The study involved 254 samples from 188 COVID-19 cases, including 43 samples at 6 to 8 months after infection. Fifty-one subjects in the study provided longitudinal blood samples, allowing for both cross-sectional and longitudinal analyses of SARS-CoV-2–specific immune memory. Antibodies against SARS-CoV-2 spike and receptor binding domain (RBD) declined moderately over 8 months, comparable to several other reports. Memory B cells against SARS-CoV-2 spike actually increased between 1 month and 8 months after infection. Memory CD8+ T cells and memory CD4+ T cells declined with an initial half-life of 3 to 5 months. This is the largest antigen-specific study to date of the four major types of immune memory for any viral infection. Among the antibody responses, spike immunoglobulin G (IgG), RBD IgG, and neutralizing antibody titers exhibited similar kinetics. Spike IgA was still present in the large majority of subjects at 6 to 8 months after infection. Among the memory B cell responses, IgG was the dominant isotype, with a minor population of IgA memory B cells. IgM memory B cells appeared to be short-lived. CD8+ T cell and CD4+ T cell memory was measured for all SARS-CoV-2 proteins. Although ~70% of individuals possessed detectable CD8+ T cell memory at 1 month after infection, that proportion declined to ~50% by 6 to 8 months after infection. For CD4+ T cell memory, 93% of subjects had detectable SARS-CoV-2 memory at 1 month after infection, and the proportion of subjects positive for CD4+ T cells (92%) remained high at 6 to 8 months after infection. SARS-CoV-2 spike-specific memory CD4+ T cells with the specialized capacity to help B cells [T follicular helper (TFH) cells] were also maintained. The different types of immune memory each had distinct kinetics, resulting in complex interrelationships between the abundance of T cell, B cell, and antibody immune memory over time. Additionally, substantially heterogeneity in memory to SARS-CoV-2 was observed. CONCLUSION: Substantial immune memory is generated after COVID-19, involving all four major types of immune memory. About 95% of subjects retained immune memory at ~6 months after infection. Circulating antibody titers were not predictive of T cell memory. Thus, simple serological tests for SARS-CoV-2 antibodies do not reflect the richness and durability of immune memory to SARS-CoV-2. This work expands our understanding of immune memory in humans. These results have implications for protective immunity against SARS-CoV-2 and recurrent COVID-19

Immunological memory to SARS-CoV-2 assessed for up to eight months after infection.

Understanding immune memory to SARS-CoV-2 is critical for improving diagnostics and vaccines, and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at ≥ 6 months post-infection. IgG to the Spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month post symptom onset. SARS-CoV-2-specific CD4 + T cells and CD8 + T cells declined with a half-life of 3-5 months. By studying antibody, memory B cell, CD4 + T cell, and CD8 + T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics

Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection.

Understanding immune memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for improving diagnostics and vaccines and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at ≥6 months after infection. Immunoglobulin G (IgG) to the spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month after symptom onset. SARS-CoV-2-specific CD4+ T cells and CD8+ T cells declined with a half-life of 3 to 5 months. By studying antibody, memory B cell, CD4+ T cell, and CD8+ T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics