Tie-ing the endothelium: development of an mRNA-based nanotherapeutic to restore microvascular barrier function for the treatment of sepsis-induced multiple organ failure

Sepsis involves a dysregulated host response to infection that can lead to life-threatening multiple organ failure. Due to the unacceptably high mortality and the lack of effective pharmacological intervention, there is an unmet clinical need to develop novel therapeutic approaches to treat sepsis. Endothelial dysfunction is a hallmark of sepsis to which the loss of Ang/Tie2 signalling, a key regulator of vascular integrity, contributes significantly, making it an attractive therapeutic target. The impairment in Tie2 pathway activity manifests most prominently in the lungs, where it drives excessive microvascular leakage and consequently can hamper organ function. Several Tie2-targeted therapeutics have shown promising preclinical efficacy in sepsis mouse models, but their clinical translation faces challenges. Most, if not all, of these strategies are dependent on the presence of Tie2. However, Tie2 availability is often compromised in sepsis patients as a consequence of transcriptional downregulation, receptor shedding, and/or genetic predisposition, which would limit clinical efficacy. The global success of the COVID-19 vaccines has showcased the use of messenger RNA (mRNA) as a therapeutic modality for transient upregulation of protein expression and precise modulation of cellular pathways. However, intracellular delivery of mRNA remains a hurdle due to its large size, polyanionic nature, and susceptibility to degradation. Lipid nanoparticles (LNPs) containing ionizable cationic lipids emerged as the most clinically advanced platform for mRNA delivery, but their application for delivery to non-liver tissues is limited. Exploring alternatives to ionizable cationic lipids, such as ionizable polymers [e.g. amino-polyesters (APE)], is therefore paramount to help expand the therapeutic utility of mRNA in acute inflammatory diseases such as sepsis. In this thesis, I explored the therapeutic potential of upregulating Tie2 in endothelial cells via synthetic mRNA delivery with the aim to protect endothelial barrier function for the prevention of sepsis-induced multiple organ failure. To this end, the thesis covers (1) the therapeutic mRNA design and validation of Tie2 mRNA to restore microvascular integrity after inflammatory injury and (2) the development of an APE-based nanocarrier that can facilitate efficient and selective mRNA delivery to endothelial cells in the lungs. In Chapter 2, the therapeutic mRNA was developed encoding the human Tie2 gene (TIE2/TEK). Several mRNA design parameters were explored to improve the Tie2 protein yield in primary endothelial cells in vitro. The inclusion of human beta-globin untranslated regions and m1 nucleoside modifications in the EGFP-tagged Tie2 mRNA led to the most robust Tie2-EGFP protein expression by up to 50-fold and 20-fold compared to the endogenous levels in HUVECs and HPMECs, respectively. Also, the introduction of missense mutations (i.e. p.I637V, p.N644H, and p.S648T) into a protease cleavage site of Tie2 reduced shedding of the Tie2 ectodomain in response to TNF-alpha, thereby maintaining receptor activity under inflammatory conditions. To validate its therapeutic efficacy, the delivery of Tie2 mRNA to HUVEC monolayers synergistically enhanced endothelial barrier integrity after ligand-mediated activation and restored TNF-alpha-induced barrier disruption. These results aligned with the morphological changes of cellular structures responsible for the integrity of the endothelial barrier, including adherens junction stabilization and cortical actin reorganization. Interestingly, expression of the protease-resistant Tie2 variant led to superior barrier-stabilising effects following prolonged exposure to TNF-alpha. Based on Moderna’s benchmark mRNA-LNP, selective Tie2 mRNA delivery to pulmonary endothelial cells in vivo was initially investigated by using a lung-targeted SM-102 LNP formulation including cationic lipid DOTAP. The cell surface expression of Tie2 was upregulated in 13% of pulmonary endothelial cells, responsible for a 25% increase in overall Tie2 expression in lung tissue. In Chapter 3, a library of 36 ionizable APEs, synthesized via ring-opening polymerization of chemically diverse tertiary amino-alcohols and lactone monomers, was investigated for its potential to facilitate efficient intracellular mRNA delivery in vitro and to target extra-hepatic tissues in vivo. Screening of these polymers formulated with lipid excipients into APE-LNPs showed that APEs containing an increasing number of tertiary amines achieved superior mRNA delivery efficacy in HUVECs. The presence of an alkyl side chain in the lactone monomer of the polymer was required for the formation of stable nanoparticles with efficient mRNA encapsulation. The length of the polymer alkyl side chain affected mRNA delivery efficacy in vitro in an amino-alcohol-dependent manner, as well as influenced tissue selectivity in vivo, with shorter side chains more effectively targeting non-liver tissues, including the lungs and spleen. By studying the role of lipid excipients in the APE-LNP formulation, the helper phospholipids were furthermore found to be imperative for mRNA delivery performance, whereas the removal of cholesterol had minimal impact. In Chapter 4, post-polymerization functionalisation of the lead APE, AA2-DL-3, was explored to improve selective mRNA delivery of APE-LNPs to endothelial cells in the lungs. The conjugation of amine-rich end-groups via degradable linkers led to the identification of an end-capped polymer, AA2-DL-3-L1-E1, with outstanding mRNA delivery efficacy comparable to commercially available transfection reagent. However, the performance of these end-capped APE-LNPs did not translate in vivo, which was linked to poor nanoparticle stability. Finally, the delivery of therapeutic Tie2 mRNA was assessed using the two top-performing APE-LNP delivery systems in vitro. The AA2-DL-3-L1-E1 LNPs facilitated the most efficient mRNA delivery in HUVECs based on Tie2-EGFP protein yield, outperforming the benchmark LNPs utilised in the marketed mRNA vaccines. However, these particular APE-LNPs impaired intracellular trafficking of the transmembrane Tie2 receptor to the cell surface as the therapeutic proteins were sequestered into lysosomal compartments. This phenomenon correlated with the induction of strong pro-inflammatory immune responses, which could be partially mitigated by the reduction of the mRNA-LNP dose. Overall, the research captured in this thesis provides initial efforts in the preclinical development of an mRNA-based nanotherapeutic targeting the endothelium for the prevention of sepsis-induced multiple organ failure. Tie2 mRNA delivery is presented as a novel therapeutic approach to restore endothelial barrier function after inflammatory injury and to complement Tie2-activating therapies that are limited to receptor availability. Also, APE-LNPs can serve as promising mRNA delivery systems for extra-hepatic tissues, but require optimization for lung-selective applications. Importantly, these results highlight emerging barriers for the successful application of APE-LNPs as an mRNA therapeutic, since nanoparticle-induced immunogenicity can diminish therapeutic efficacy and raise safety concerns in diseases with underlying inflammation, such as sepsis. Collectively, these studies underscore the importance of tailoring both the therapeutic mRNA and the nanocarrier to support their intended therapeutic use.

Grantor dc:publisher

University College Cork

Year dc:date.issued

2025

Author dc:creator

• Mulder, Lianne M.

Advisors dc:contributor.advisor

• Kowalski, Piotr
• Ryan, Katie

• Sepsis
• Multiple organ failure
• Vascular permeability
• Endothelial barrier dysfunction
• Tie2 receptor signaling
• Messenger RNA
• mRNA therapeutics
• mRNA delivery
• Lipid nanoparticles
• Polymeric nanoparticles
• Amino-polyesters
• Endothelial cell targeting
• Lung selectivity
• Inflammation

Statement dc:rights

• © 2025, Lianne Mulder.

Licence dc:rights.uri

• https://creativecommons.org/licenses/by-nc/4.0/

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10468/18926

OAI identifier oai:identifier

oai:cora.ucc.ie:10468/18926