Silencing the Storm: Stem Cell-Derived Extracellular Vesicles as a Novel Therapeutic for Neuroinflammation

Abstract

Chronic neuroinflammation, defined as immune overactivation within the central nervous system (CNS), is a key driver of many neurodegenerative diseases. Yet current therapies inadequately modulate it to improve prognosis. In GM2 gangliosidosis, a fatal lysosomal storage disorder marked by rapid neurodegeneration and progressive neuroinflammation, inflammation persists even after gene therapy resolves the primary etiology, ultimately undermining sustained efficacy. Addressing chronic neuroinflammation is therefore essential to improving outcomes in neurodegenerative diseases and GM2 gangliosidosis. This dissertation investigates the therapeutic potential of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs), specifically EVs from MSCs cultured under inflammatory conditions (IFEVs), as a novel anti-inflammatory strategy, using an animal model of feline GM2 gangliosidosis. Chapter 2 establishes an in vitro platform for feline neuroinflammation to demonstrate the immunomodulatory properties of MSC-EVs and to identify the culture conditions that yield the most potent EVs, since EV functionality depends on parent-cell conditions. MSC-EVs suppressed pro-inflammatory factors in lipopolysaccharidestimulated feline glia, and IFEVs showed enhanced efficacy over other EV isolates. Chapters 3 and 4 translate IFEVs' anti-inflammatory properties, in vitro and in vivo, into the naturally occurring feline model of GM2 gangliosidosis. 2 Chapter 3 found that IFEVs exhibited anti-inflammatory effects in the CNS and potentially in the periphery. In inflammatory glia from GM2 cats, pro-inflammatory cytokines (IL-6, TNF-α, IL-1β) were reduced after 48 hours by downregulating NF-κB and NLRP3 inflammasome signaling and upregulating arginase 1. IFEVs may also promote systemic immune homeostasis by increasing regulatory T-cell proliferation in vitro. Chapter 4 is the first in vivo demonstration of IFEV therapeutic properties in a large-animal neurodegenerative model. Repeated intravenous administration in presymptomatic GM2-affected cats was safe and reduced neuroinflammation by downregulating pro-inflammatory mediators (TNF-α, IL-1β, p65, NLRP3) and attenuating CNS gliosis. Transcriptomic analysis confirmed a robust neuroinflammatory signature in GM2, with IFEVs showing limited reversal. Though IFEVs did not correct the enzymatic deficiency or significantly alter clinical biomarkers, their potent anti-inflammatory effects could complement gene therapy. This dissertation demonstrates that stem cell-derived EVs, particularly IFEVs, modulate inflammatory responses in GM2 gangliosidosis and could extend to other neurodegenerative diseases with persistent neuroinflammation, ultimately improving prognosis.