Mina Yeganeh, BSc - Best of the Best in Pediatric Surgery 2024

Awesome. So, we are ready for the 3rd heat of the day, and for that, we have BAPS, that is the British Association of Pediatric Surgery, and that includes for the 1st time this year, so we are glad to have them. And we have UUSA, the European Pediatric Surgeons Association, and PAPS. So, let's start with the first one, that's from UUSA, Doctor Mina. Yegane representing um Usa with lipid nanoparticle delivery of microRNA um attenuates intestinal inflammation during um an experiment. So let's hear it. Hi everyone, my name is Mina. I'm a master's student in Dr. Agostino Piro's lab at Sicketts Hospital in Toronto. Thank you for this opportunity to present our work here today regarding nucleic acid-based approach for the treatment of experimental necrotizing enterocolitis. To start off, necrotizing enterocolitis, or neck is a devastating gastrointestinal disease in neonates. It starts with severe intestinal inflammation, which can lead to intestinal necrosis and perforation and ultimately multi-system organ failure. Now, in previous years, our lab has shown that administration of human milk extracellular vesicles, or EVs, protects mice from developing neck. As you can see here on the right, mice that received milk EVs in blue have significantly lower intestinal injury scores, as well as lower intestinal inflammation compared to untreated neck. Despite these protective effects, the underlying mechanisms of milk EVs remain unclear, preventing their clinical translation. Therefore, we wanted to look at factors within milk EVs that contribute to these protective effects, and we found that microRNA 148A, which is the most abundant microRNA in milk EVs, has shown to reduce the inflammatory response in other disease models. This anti-inflammatory effect of neuron48A is mainly known to be through the inhibition of T-like receptor 4 or TLR-4 pathway. TLR-4 is a pattern recognition receptor that is critical for the initiation for inflammatory response, and a lot of the work from Doctor David Hackam's group has shown that TLR4 signaling is actually required for the development of experimental neck. Therefore, the inhibition of this pathway offers opportunities for therapeutic intervention in neck. The aim of this study was therefore to investigate the potential therapeutic role of macaroni 148 in the intestine during experimental neck. And we used a well established mouse model of neck where neck is induced from postnasal day 5 or P5 to P9 by gavash feeding of hyposmolar formula, lipopolysaccharide, as well as exposure to intermittent hypoxia. And on day 6 and 7, we administered either mRNA 1488 or a TLR4 silencing RNA antagonist, which acts as a positive control in our studies, and both of these nucleic acids were given by enema. To deliver these nucleic acids to the intestine, we employed a lipid nanoparticle or LNP delivery system. These LNPs encapsulate the nucleic acids and release them inside the cells in a pH responsive manner. We first administered the LMPs in healthy pups through enema, and after a couple hours, my 148A, which was fluorescently tagged, was present in the cytosol of the intestinal epithelial cells indicated here by the green signal. This is, this is showing that the LMPs successfully delivered the nucleic acids to the intestinal epithelium in these uh neonatal muses. We then administered microRNA 148A and the TLR4 siRNA antagonist in the experimental neck model. You can see that neck results in a significantly higher injury rate compared to the control mice, and the administration of microRNA 148A and the TLR 4 siRNA both significantly reduced this injury. Next, we looked at the expression of uh pro-inflammatory cytokines TNF alpha and IL-6 in the intestine. Again, as expected, you can see a significant increase in gene expression of these pro-inflammatory cytokines during your neck, compared to the control mice, while the administration of muron48A and the TLR-4 antagonists both result in significantly lower inflammation in the intestine. And lastly, we use a reporter cell line that allows us to measure the activity of NF kappa V, which is a transcription factor downstream of TLR 4, and you can see that the control cells in green showed very little NF kappa B activity on the Y axis. When we administered lipopolysaccharide or LPS, it activates the t-like receptor pathway, and we see a significant rise in NF kappa B expression in red. We then administered LPS plus either MR 148A or the TLR-4 antagonist at two different concentrations, and you can see that these nucleic acids both significantly lower the NF kappa B expression at both concentrations. In conclusion, we have shown that the lipid nanoparticles successfully delivered the MR 14A to the distal ileum of neonatal mice, and during experimental neck, um, administration of MR148A results in reduced intestinal injury and inflammation, and this is mainly through the downregulation of NF kappa B signaling. And with that, I'd like to thank the Pure Lab for their constant support and thank you for listening. I'd be happy to take any questions. Awesome. Um, well, Doctor Yegane, thank you for this presentation. I really enjoy it. I have only one question. Is that if this microRNA can only palliate the injury or can we treat neck in a preventive way? That's a great question. Thank you. Um, it's really hard to distinguish between preventative and therapeutic, uh, interventions with the, uh, experimental model that we have right now for neck. Um, it's a very short model. It starts from P5 to P9. And we see the surge in intestinal injury and inflammation around P6. So it starts and it sort of increases from P6 to P7. And that's when we try to have our therapeutic, um, intervention. And that sort of time span to, um, to sort of count as a therapeutic measure. But if you want to start earlier on P5, it would be acting as a preventative measure. So right now, in this study, we looked at it as a therapeutic approach, but it's, it's very difficult to, to distinguish between those two with the current model. Awesome, um, thank you, thank you very much. Let's see, uh, then if we have, uh, some questions in the chat, you can always answer from there, and, uh, we share. Thank you so much. Thank you. Thank you so much. Great work.