Targeted gene delivery using ultrasound (US) and microbubbles (MB) is in development as a clinically applicable gene therapy (GT) technique. There is limited research targeting the placenta, which is a potential therapeutic strategy for preeclampsia (PE) since PE is a complex genetic disorder with ineffective management strategies. Differentially regulated placental microRNAs (miRNAs) in PE may represent suitable targets for GT. The aims of the study were 1) to develop an US and MB gene delivery protocol, with applicability to the placenta, by conducting a proof-of-concept study to demonstrate site-specific gene transfection and 2) to identify differentially expressed placental miRNAs in 3rd trimester PE patients, through a systematic literature review, and conduct a comparison of expression in a PE rodent model recently published by our group (1). Female CD1 mice were anaesthetised with 5% isoflurane and maintained on 1.5% isoflurane during procedures. MB (SonoVue) and plasmid (PGL3) were administered systemically to treatment mice (n=3), followed by exposure of the heart to US (B-mode, H14, 1.8 M.I., 1cm focal depth, 2 minutes), using Siemens Acuson Sequoia-512 system and 15L8 probe. Plasmid was injected intramuscularly to mouse gracilis muscle as a positive control (n=1). Luciferase assay was performed and normalised to protein concentration measured by Pierce BCA protein assay to evaluate gene transfection; values are mean CPS/µg of protein±SD (n=4 technical replicates per tissue). Significantly differentially expressed placental miRNAs in PE patients were identified as candidate miRNAs based on detection by ≥3 screening studies. Expression of candidate miRNAs was measured by qRT-PCR in PE rat model placentas; values are mean RQ (RQ min-max) vs. non-PE controls (n=4 biological replicates per group). Luciferase activity was detected in the gracilis muscle (6.17±4.51 CPS/µg of protein) of the positive control mouse. Luciferase activity was detected in the atria of treatment mouse 2 (14.6±19.5 CPS/µg of protein) and ventricles of treatment mouse 3 (6.43±5.84 CPS/µg of protein), providing evidence of site-specific gene transfection. Less luciferase activity was detected in the atria and ventricles of treatment mouse 1 (3.52±1.95 and 1.04±0.20 CPS/µg of protein, respectively). The systematic literature review identified nine candidate miRNAs. MiR-223 and miR-181a were significantly differentially expressed in PE rat model placentas (RQ: 1.46(0.12-0.13)-fold upregulation and 0.81(0.05-0.05)-fold downregulation, respectively, p<0.05, unpaired t-test). MiR-223 and miR-181a, which have been shown to play roles in trophoblast migration and invasion (2) (3), represent targets for US and MB gene delivery. Future studies will optimise the US and MB gene delivery protocol for translation to targeting the placenta in our PE rodent model, a novel application of the GT technique.
|Publication status||Published - 2019|
|Event||Physiology 2019: Placental and Perinatal Physiology: Proceedings of the Physiological Society 43, PC262 - Aberdeen|
Duration: 8 Jul 2019 → 10 Jul 2019
|Conference||Physiology 2019: Placental and Perinatal Physiology|
|Period||8/07/19 → 10/07/19|
- altered placental microRNAs
- gene therapy