2024 American Thoracic Society BEAR Cage Winning Proposal: Endothelial-targeted mRNA Lipid Nanoparticles for Acute Respiratory Distress Syndrome

Now entering its 11th year, the BEAR Cage (Building Education to Advance Research) is an annual competition that provides American Thoracic Society (ATS) awards to new or early-stage investigators to support innovative research projects. Finalists present their ideas to a panel of translational science experts at the ATS International Conference. The 2024 winning proposal is described in this editorial.

Acute respiratory distress syndrome (ARDS) is a life-threatening disease that is characterized by acute alveolar inflammation and innate immune cell activation, resulting in alveolar epithelial and endothelial barrier disruption. These series of events manifest clinically as noncardiogenic pulmonary edema and respiratory failure requiring mechanical ventilation (1). ARDS burden is high, affecting about 190,000 patients annually in the United States, with a mortality rate of 40% (2). Despite the progress made over the past decade in understanding ARDS pathophysiology, optimizing supportive treatment, and identifying disease subphenotypes, no new therapies have been successfully translated clinically (3).

ARDS therapeutics have failed clinical trials because ARDS is a heterogeneous disease with multiple pathways, some of which have no known small molecule inhibitors. Also, ARDS is a critical illness that often affects patients with multiorgan dysfunction, thus increasing susceptibility to systemic drug off-target effects. Therefore, to develop a successful new therapeutic for ARDS, the drug will need to be able to concentrate significantly in the lungs to minimize off-target effects, and appropriately target multiple aberrant pathways involved in the ARDS pathophysiology. Inhaled delivery of drugs for ARDS could improve organ-specific drug delivery; however, it is limited by the influx of immune cells into the alveolar space that phagocytose drugs and the significant pulmonary edema that impairs drug absorption (4).

Thus, we propose a nanoparticle-based therapy that targets the pulmonary endothelial cell via intravenous administration. We are targeting the endothelium because it is a key cell type involved in ARDS pathophysiology and can be easily accessed via the intravenous delivery route, thus creating a reasonable alternative to the limitations of inhaled drugs for ARDS. The nanoparticle will encapsulate an mRNA cargo that expresses a therapeutic protein, facilitating the targeting of challenging pathophysiological pathways. The feasibility of mRNA lipid nanoparticle (LNP)-based therapeutics has already been demonstrated by the success of the coronavirus disease (COVID-19) mRNA vaccine (5, 6). Also, this platform technology will ease the development of personalized, phenotype-specific therapies, thus addressing the heterogeneity of ARDS.

The Solution

Endothelial-targeted nanoparticles comprise the following: a lipid nanoparticle, mRNA cargo, and affinity ligands specific to the endothelial CAMs (cellular adhesion molecules) (Figure 1). Lipid nanoparticles are nanoscale carriers, measuring <200 nm and composed of ionizable lipids, phospholipids, cholesterol, and polyethylene glycol lipids, all of which contribute to stability, biodistribution, and encapsulation of the RNA and successful cargo release during administration in vivo. To achieve lung-specific uptake, mRNA lipid nanoparticles are coated with antibodies on the surface that can bind to endothelial CAMs such as PECAM (platelet-derived endothelial CAM) (Figure 2). When delivered intravenously, these particles accumulate primarily in the lungs, at 300-fold higher concentration than typical small-molecule drugs delivered intravenously (7, 8). This is because the intravenous route allows first-pass delivery to the lungs, and the lungs’ extensive capillary network receives the entire cardiac output (7, 8).

Figure 1.

mRNA lipid nanoparticle. Figure 1 created with BioRender.com.

Figure 2.

Mechanism of endothelial target nanoparticle uptake and mRNA cargo expression. Figure 2 created with BioRender.com.

Preclinical Studies on Endothelial-targeted LNPs

As we develop our nanotherapeutic, we have encountered a few challenges. We have shown that our endothelial-targeted nanoparticles are able to achieve lung-specific uptake. However, using flow cytometry to study cell-type specificity of nanoparticle uptake, we showed that the organ specificity was driven not only by endothelial cell uptake but also by uptake by marginated neutrophils (9). Marginated neutrophils are resident neutrophils in the lung capillaries that play a role in pathogen surveillance and form a key portion of the lung innate immune system (10, 11). We showed that these neutrophils recognize the nanoparticles via several mechanisms, such as Fc recognition of our conjugated antibodies. When we modified the Fc portion of our antibodies, we were able to achieve more endothelial-specific cellular uptake and decrease neutrophil uptake (9). Therefore, we addressed a technological hurdle to improve endothelial cell specificity, as the endothelial cells are the nexus of pulmonary pathophysiology in diseases like ARDS.

In addition to optimizing our cell-specific targeting, we have also explored various therapeutic cargos for ARDS. One specific promising candidate for LNP cargo is the IL10 mRNA (12). IL10 is a cytokine known to have antiinflammatory properties, and, thus, we sought to evaluate it as a candidate to diminish inflammation in ARDS. Using nebulized LPS as a mouse model for ARDS, we administered IL10 mRNA LNPs and, 24 hours later, killed the mice and collected their BAL fluid for protein measurement and leukocyte count. We showed that there is decreased leukocytosis in the BAL fluid at 24 hours; however, there was no significant effect on protein concentration. This therefore suggests that we are only partially ameliorating ARDS in the current therapeutic model.

Further Directions

Having identified the challenges in our nanoparticles, we now propose solutions that encompass several next steps. First, we will increase the endothelial cell specificity of our nanocarriers using Fc-absent antibodies for endothelial targeting. This will include affinity ligands such as Fab or F(ab)2′ fragments. Second, we will optimize our mRNA cargo. Currently, our IL10 mRNA only targets acute inflammation in ARDS and does not address ongoing endothelial leak and thus alveolar fluid accumulation. Therefore, we will compare IL10 cargo to other cargos that express proteins that are involved in endothelial cell junctions and cytoskeletal stabilization. This approach will help mitigate endothelial cell leakiness, thereby reducing pulmonary edema. Overall, it establishes a comprehensive therapeutic strategy to address important pathogenic mechanisms of ARDS.

Beyond ARDS, the endothelial-targeted mRNA LNP is a versatile platform technology, with potential applications in other acute lung injury syndromes, such as primary graft dysfunction in lung transplantation and pulmonary vascular diseases. Therefore, the results from this project will in addition pave the way for novel treatments for other pulmonary vascular conditions driven by endothelial cell dysfunction.

Conclusions

Endothelial-targeted mRNA LNPs present a promising therapeutic for treating ARDS. With the support from the ATS BEAR Cage Award and the ATS Quad D committee we will be able to develop a therapeutic that will successfully reach patients with ARDS.