Label‐free quantitative proteomic analysis of extracellular vesicles released from fibroblasts derived from patients with spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that represents a significant cause of infant mortality. SMA is characterized by reduced levels of the Survival Motor Neuron protein leading to the loss of alpha motor neurons in the spinal cord and brain stem as well as defects in peripheral tissues such as skeletal muscle and liver. With progress in promising therapies such as antisense oligonucleotide and gene replacement, there remains a need to better understand disease subtypes and develop biomarkers for improved diagnostics and therapeutic monitoring. In this study, we have examined the utility of extracellular vesicles as a source of biomarker discovery in patient-derived fibroblast cells. Proteome examination utilizing data-independent acquisition and ion mobility mass spectrometry identified 684 protein groups present in all biological replicates tested. Label-free quantitative analysis identified 116 statistically significant protein alterations compared to control cells, including several known SMA biomarkers. Protein level differences were also observed in regulators of Wnt signaling and Cajal bodies. Finally, levels of insulin growth factor binding protein-3 were validated as being significantly higher in extracellular vesicles isolated from SMA cells. We conclude that extracellular vesicles represent a promising source for SMA biomarker discovery as well as a relevant constituent for advancing our understanding of SMA pathophysiology.

Bioengineering extracellular vesicle cargo for optimal therapeutic efficiency

“…EV cargo content usually reflects the state of the cell from which they originate, and simply overexpressing a protein of interest within donor cells can lead to release of EVs loaded with high levels of that protein. 103 , 104 For example, we showed that plasmid- or adenovirus-mediated overexpression of survival motor neuron (SMN) protein in donor cells led to release of exosomes with elevated concentrations of SMN protein. 83 Countless studies have shown a similar effect with numerous different proteins.…”

Molecular Biomarkers for the Diagnosis, Prognosis, and Pharmacodynamics of Spinal Muscular Atrophy

“…Bianchi et al analyzed the whole proteome in the CSF of 10 SMA 1 patients and 7 healthy controls (HCs) and observed 39 differentially expressed proteins between SMA patients and HCs (with APOA1, hemoglobin subunit β, hemoglobin subunit α, and transthyretin being the most significant) [48]. In extracellular vesicles released from fibroblasts of one SMA 1, two SMA 2, and three HC subjects, Roberto et al observed 116 differentially expressed proteins (with IGFBP3, Plastin 3, PTK7, TCP1, FETUA, and FXA being the most significant) [49]. Kobayashi et al analyzed nearly 1000 plasma proteins in 266 SMA patients and 22 HCs.…”