PROJECT SUMMARY Lung cancer remains the most lethal cancer among both women and men in the United States. There is a clear need for developing new and more effective therapeutics for the treatment of lung cancer, especially the most predominant subtype non-small cell lung cancer (NSCLC). As posttranscriptional gene regulators, the genome-derived microRNAs (miRNAs or miRs) govern many critical cancer cellular processes such as proliferation, invasion, and stemness. Therefore, restoration of tumor suppressive miRNAs (e.g., miR-124-3p (miR-124)) lost or downregulated in NSCLC cells represents a novel therapeutic approach. However, current research relies primarily on the use of chemo-engineered miRNA mimics (ChemoRNAs) synthesized in vitro and comprised of extensive and diverse types of artificial modifications at various locations, which are different from natural RNA molecules produced and tolerated in vivo that do not contain any modifications or just a few necessary posttranscriptional modifications. Our past and ongoing efforts have led to a patented platform technology to achieve a robust, high-yield, and large-scale in vivo fermentation production and use of true biologic or bioengineered RNA agents (BioRNAs). We have demonstrated that BioRNAs, consisting of only several natural modifications, are precisely processed to target miRNAs (or siRNAs) to selectively regulate target gene expression in human NSCLC cells and subsequently, inhibit NSCLC cell proliferation and invasiveness as well as tumor progression and metastasis. Among the NSCLC-relevant biologic RNAs, we have identified a few lead miRNAs (e.g., miR-124 and miR-22-3p (miR-22)) consistently exhibiting potent antiproliferative activities against multiple NSCLC cell lines. Our preliminary studies have also showed that BioRNA/miR-124 is more efficacious than commercial ChemoRNA/miR-124 to regulate target gene expression and NSCLC cell viability, and miR-124 may control NSCLC immunity via modulating specific immune regulator and checkpoint protein levels. Further, we have found that miR-22 controls NSCLC metabolism through the regulation of key nutrient metabolic enzymes and transporters. Moreover, humanized BioRNA/miR-124 and miR-22 lipopolyplex monotherapy effectively reduced tumor progression in animal models without causing any hepatic or renal toxicity or severe immunogenic effects. Given such exciting preliminary findings, we hypothesize that novel bioengineered RNA molecules can be utilized to modulate NSCLC metabolism and immunity to improve therapeutic outcomes of current medications via pharmacodynamic and pharmacokinetic interactions. To test the hypothesis, we propose to define the biosimilarities between model Bio- and Chemo-RNA molecules by side-by-side comparing their efficacy and specificity in the regulation of target gene expression and inhibition of NSCLC cell growth (Aim 1), delineate the molecular pharmacological actions of lead BioRNAs in the control of NSCLC cell m...