Bioengineering of Enucleated Cell Therapeutics for Treating Inflammatory Diseases

New therapeutics often fail to reach the clinic due to poor efficacy and severe adverse toxicity, primarily arising from inadequate drug delivery mechanisms. This inefficiency costs the pharmaceutical industry billions annually, underscoring the urgent need for targeted drug delivery systems that can accurately target diseased tissues while minimizing off-target toxicity. In recent years, innovative delivery strategies—such as nanoparticles, exosomes, whole cells, and red blood cells— have been developed to deliver therapeutic agents. However, despite these advancements, no fully developed and clinically viable intravenous drug delivery system currently exists. Thus, the development of precise delivery mechanisms to diseased tissues offers a significant opportunity to treat a broad spectrum of conditions safely. Our laboratory has pioneered a novel therapeutic delivery system, "Cargocytes," designed to transport therapeutic cargo directly to inflamed and damaged tissues with precision. These transporters are derived from mesenchymal stem cells (MSCs) genetically engineered with chemoattractant receptors (CCR2 and CXCR4) and the endothelial adhesion molecule (PSGL-1). This GPS-like guidance system allows Cargocytes to respond to P- and E-selectins on inflamed endothelial cells and chemoattractants (CCL2 and CXCL12) secreted by diseased tissues. The MSCs are then enucleated via gentle density gradient centrifugation and transfected with mRNAs encoding the therapeutic payload before intravenous administration. Our work shows that enucleation bestows the Cargocyte with unique functional and biophysical properties while providing significant safety advantages for therapeutic delivery. This application aims to: Aim 1: Optimize the in vitro production, cryopreservation, and biobanking of Cargocytes for subsequent studies. Aim 2: Conduct comprehensive immunogenicity and organ toxicity tests in vitro and in vivo using established immunocompetent mouse models. Aim 3: Evaluate the ability of optimized Cargocytes carrying an IL-10 payload to treat inflammatory pancreatitis effectively and safely in a preclinical mouse model of caerulein-induced chronic pancreatitis. Through these objectives, we aim to establish Cargocytes as a groundbreaking therapeutic delivery platform capable of addressing the unmet need for precise, targeted drug delivery in clinical applications.