PROJECT SUMMARY New strategies to combat pneumonia caused by different pathogens are urgently needed. Inflammatory macrophages play an essential role in clearing bacteria, fungi, and viruses during infections; however, hyperinflammatory responses mediated by these cells can cause severe side effects, including death. Immunotherapy that modulates macrophage polarization has shown promise in suppressing hyperinflammatory responses while retaining the capability of macrophages to clear pathogens. To achieve successful immunotherapy with RNA therapeutics, the following obstacles must be overcome: (1) rapid clearance of RNA therapeutics by RNase in tissues; (2) poor cellular uptake of free RNA therapeutics; and (3) loss of RNA therapeutics to non-infected tissues and potential off-target side effects. We previously discovered and successfully transitioned to industry two nanotherapeutic systems that addressed these limitations for bacterial infections. The elements of the nanosystems relevant to the present proposal are: high loading capacity for RNA therapeutics; an ability to protect the RNA payload from degradation in vivo; and highly selective targeting of the macrophages via pendant peptides. The proposed project hypothesizes that this approach may be generally applicable across a spectrum of pathogens, and it aims to investigate treatment of viral and fungal pulmonary infections. To address these goals, a deeper understanding of macrophage function and nanoparticle interactions is needed, particularly in the context of pathogenic infections. Through three Specific Aims, we propose to optimize and then evaluate three major nanoplatform-based systems that have shown promise for nucleic acid delivery, and investigate the in vivo biological interactions of the targeted nanoplatforms to obtain deeper understanding of macrophage polarization in combating pulmonary infections: (1) Develop a targeting strategy for macrophage homing in infected lungs. We hypothesize designs that will allow the nanoparticle to reach the infected regions of the lungs while preserving the potency of the RNAi therapeutic, either by i.v. or by direct pulmonary delivery of nanoplatforms. This Aim will focus on screening for new peptides that target macrophages in lung infection models, using the existing macrophage-targeting peptide CRV as a benchmark. We will focus on well-established mouse models of pneumonia induced by carbapenem-resistant K. pneumoniae, A. fumigatus, and influenza A. (2) Develop, evaluate, and then downselect from three broad classes of nanoplatforms (i.e., lipid nanoparticles, tandem peptide nanoparticles, and fusogenic porous silicon nanoparticles) to load RNA therapeutics. These nanoplatforms will be targeted to macrophages in the infected lungs using peptides from Aim 1. Cytotoxicity, gene knockdown efficiency, and macrophage polarization will be evaluated in vitro. Pharmacokinetics including macrophage targeting and tissue distribution will be studied...