Developing Novel ALOX5 mRNA Nanoparticles To Treat Incurable Non-Small Cell Lung Cancer

<p dir="ltr">Lung cancer (LC) remains one of the most prevalent and deadliest malignancies worldwide, with over 2.2 million new cases and 1.8 million deaths reported in 2020. In Australia, LC ranks among the top five causes of death and imposes a significant economic burden. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all LC diagnoses and is associated with poor prognosis, with five year survival rates below 25%. The two major NSCLC histological subtypes comprise lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD), which differ in cellular origin, anatomical location, and histopathological features. While cigarette smoking, environmental exposures (e.g., air pollution) and genetic mutations (e.g., oncogene activation and tumour suppressor gene inactivation) are established contributors to LC, chronic inflammation has recently emerged as a key factor in disease progression. The accumulation of tumour associated neutrophils (TANs) and tumour associated macrophages (TAMs) promote inflammation which can support tumour growth and immune suppression. This thesis investigates the inflammatory landscape of NSCLC and explores arachidonate 5-lipoxygenase (ALOX5), a gene encoding the 5-lipoxygenase (5-LO) enzyme expressed in leukocytes as a potential therapeutic target. </p><p dir="ltr">To explore the role of ALOX5 in the tumour microenvironment (TME), a cohort of LUAD, LUSC and control lung tissue were analysed using immunohistochemistry, immunofluorescence and reverse transcription PCR. ALOX5 mRNA was significantly downregulated in tumour tissue, with the most pronounced reduction in LUSC. CD68+ macrophages were identified as the predominant source of 5 LO within the TME, with significant infiltration of macrophages observed in LUSC biospecimens. Similarly, there was a significant influx of MPO+ neutrophils in both NSCLC subtypes, positively correlating with classic neutrophil chemokines IL-8 and C-X-C motif chemokine ligand (CXCL)-1 expression. Although CD8+ T cell numbers were elevated in tumour biospecimens, the neutrophil-to lymphocyte ratio (NLR) was significantly increased and negatively correlated with ALOX5 expression. These findings suggest that ALOX5 downregulation may promote neutrophil accumulation and cytotoxic T cell suppression, reinforcing inflammation in the TME and positioning ALOX5 as a potential biomarker for immune imbalance. </p><p dir="ltr">Given the suppression of ALOX5 in NSCLC and its predominant expression in macrophages, we developed a lipid nanoparticle (LNP)-based mRNA delivery platform to target these cells. Our LNP library was formulated with the helper lipid monoolein (MO), in combination with cationic ionisable lipids SM-102 (SM) or ALC-0315 (ALC). LNPs were tested in immortalised mouse alveolar macrophages (MH-S), loaded with enhanced green fluorescent protein (EGFP) mRNA and labelled with Cy5 dye, with cell localisation and mRNA transfection quantified via flow cytometry. LNPs formulated with equal parts, 50 mol% MO and SM or ALC were not toxic to MH-S macrophages and displayed the strongest cellular association. However, only the SM/MO formulation produced detectable GFP fluorescence signal, attributed to its gyroid cubic (Q2G) phase, which we have identified as the optimal structure for macrophage transfection. We further enhanced the efficacy of our LNPs by formulating them with the addition of cholesterol, where 20 mol% cholesterol (CHL20) increased MH-S transfection efficiency to 70%. While higher cholesterol content reduced transfection in MH-S macrophages, 40 mol% cholesterol LNPs (CHL40) proved optimal for transfecting primary monocyte derived macrophages, yielding two novel LNP formulations optimised for mRNA delivery to immortalised and primary macrophages. </p><p dir="ltr">As we have identified ALOX5 to be supressed in NSCLC and developed a LNP formulation that successfully transfects macrophages, we proceeded to determine if we can transfect macrophages within the lung in mice. To assess in-vivo delivery, LNPs were administered intravenously (IV) or intranasally (IN) in mice. IV-delivered LNPs localised to multiple organs: lung, heart, liver, spleen and kidneys, but transfection was limited to the liver and spleen. IN delivered CHL40 LNPs remained immunologically inert and was restricted to the lungs of treated mice, efficiently bypassing the upper airways. CHL40 and Pfizer LNPs achieved efficient lung transfection, producing diffuse luciferase activity across the entire tissue, with CHL40 LNPs localising with 75% of bronchoalveolar lavage (BAL) cells in treated mice. With this work we have identified novel LNP formulations that can transfect specific organs according to the administration method and lipid composition. </p><p dir="ltr">In summary, this thesis identified that ALOX5 expression is markedly reduced in lung tumours, and inversely associated with TANs and the NLR, implicating this gene as an important regulator of the TME in NSCLC. We propose that ALOX5 is both a biomarker and therapeutic target. We confirmed that macrophages were the main source of 5-LO in the TME, therefore we optimised novel LNPs capable of delivering mRNA payloads to immortalised and primary macrophages in-vitro. We built upon this work with targeted transfection of lung tissue using our LNPs. This work provides foundational evidence for reconstituting ALOX5 expression in TAMs via the application of novel LNP formulations as a potential strategy for treating NSCLC.</p>