Extracellular Vesicle-Mediated Delivery of Survival Motor Neuron Protein: A Potential Therapeutic Strategy for Spinal Muscular Atrophy

Abstract

Spinal Muscular Atrophy (SMA) is a genetic neuromuscular disorder caused by homozygous mutation or deletion of the survival motor neuron 1 (SMN1) gene, leading to a low quantity of SMN protein in cells. This depletion of SMN protein preferentially leads to death of motor neurons and, consequently, muscle atrophy, in addition to defects in many other peripheral tissues. Although several disease-modifying therapies for SMA exist, there is no cure, and new therapies need to be developed. SMN protein is naturally loaded into extracellular vesicles (EVs), which are sub-micron-sized, membrane-bound particles released from all cell types and found in all bodily fluids. The innate ability of EVs to deliver cargo to recipient cells has caused these vesicles to gain interest as therapeutic delivery vehicles. In this thesis, I explore EVs as a delivery vehicle of SMN protein. The work presented herein demonstrates that adenovirus-mediated overexpression of SMN protein in HepG2 cells leads to the release of EVs loaded with high levels of SMN protein into conditioned medium. Application of this medium to recipient cells in tissue culture led to uptake of the SMN protein, which subsequently transited to the nucleus and co-localized with Gemin2 protein, forming nuclear gem-like structures similar to the native SMN protein. This work demonstrates that SMN protein can be delivered to cells through EVs, however SMN protein-loaded EVs would likely need to be administered on a daily basis to maintain a therapeutic threshold of SMN protein. Thus, I built upon this work by utilizing a gene therapy approach to engineer the liver of mice to release extracellular vesicles enriched with SMN protein, facilitating systemic distribution of the protein throughout the animal. The findings contained within this thesis demonstrate a viable approach to achieve body-wide distribution of SMN protein, which could be utilized as a potential therapeutic strategy to treat SMA. This approach could be used to deliver numerous therapeutic cargoes, holding promise as a potential therapy for patients presenting with diverse diseases.