Extracellular vesicles efficiently deliver survival motor neuron protein to cells in culture

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. SMN protein is naturally loaded into extracellular vesicles (EVs), which are sub-micron-sized, membrane-bound particles released from all cell types. The innate ability of EVs to deliver cargo to recipient cells has caused these vesicles to gain interest as therapeutic delivery vehicles. In this study, we show 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. Overall, this work demonstrates that SMN protein can be delivered to cells through EVs, which holds promise as a potential therapy for patients with SMA.

Keywords: Extracellular vesicles; Neuromuscular disease; Nuclear gems; Spinal muscular atrophy; Therapeutic delivery; Therapeutic protein.

Fig. 1

The presence of a 3xFLAG-tag on SMN protein does not affect protein localization within the cell or ability to interact with normal cellular protein partners. Panel A: A HepG2 cell line stably expressing a 3xFLAG-tagged SMN protein was assessed for protein expression by immunoblot. Grey arrows indicate the 3F-SMN protein and the black arrow indicates endogenous SMN protein. Panel B: Co-immunoprecipitation (Co-IP) was performed using anti-FLAG or isotype control (IgG). Input, bound, and unbound fractions were assessed by immunoblot. Grey arrows indicate the 3F-SMN protein and the black arrow indicates endogenous SMN protein. Panel C: Epifluorescence microscopy images of HepG2:3F-SMN cells stained with antibodies to tubulin and SMN, or tubulin and FLAG. Arrows indicate representative nuclear gems. Scale bars represent 10 µm. Panel D: Quantified nuclear gems per 100 cells in epifluorescence microscopy images of HepG2 and HepG2:3F-SMN stained with anti-tubulin and anti-SMN. Data was tested for significance using an unpaired t-test, where two asterisk (**) indicates p < 0.01. Data represents 3 independent experiments.

Fig. 2

EVs released by HepG2:3F-SMN cells contain 3F-SMN protein. Panel A: Conditioned medium (CM) was collected from HepG2 and HepG2:3F-SMN cells 72 h post-plating. Cellular debris and apoptotic bodies were removed by low-speed centrifugation, and EVs were isolated by ultracentrifugation. Particle diameter was assessed by nanoparticle tracking analysis. Panel B: Protein content of isolated EVs was examined by immunoblot with the indicated antibodies, with total cellular lysates from the two cell lines included as controls.

Fig. 3

Generation and characterization of an A549 cell line expressing an HA-tagged Gemin2 protein. Panel A: An A549 cell line stably expressing HA-tagged Gemin2 protein was assessed by immunoblot with the indicated antibodies. Panel B: Co-immunoprecipitation (Co-IP) was performed using anti-HA or isotype control (IgG). Input, bound, and unbound fractions were assessed by immunoblot. Panel C: Epifluorescence microscopy images of A549:HA-Gemin2 cells stained with anti-HA and anti-Gemin2. Arrows indicate nuclear gems. Panel D: Epifluorescence microscopy images of A549:HA-Gemin2 cells stained with anti-HA and anti-SMN, with arrows indicating nuclear gem-like structures. Scale bars represent 10 µm. Panel E: Quantified SMN-positive nuclear gems per 100 cells in epifluorescence microscopy images as processed in panel D. Data was tested for significance using an unpaired t-test, where two asterisk (**) indicates p < 0.01. Data represents 3 independent experiments.

Fig. 4

Adenoviral-mediated overexpression of 3F-SMN protein in HepG2 cells leads to the release of EVs containing high levels of the 3F-SMN protein. Panel A: HepG2 cells were infected with Ad3F-SMN at an MOI = 50, 100, 200, or mock infected (PBS). Seventy-two hpi, cell lysates were collected and EVs were isolated using polyethylene glycol (PEG). Protein content of the EVs was examined by immunoblot. Endogenous SMN protein is indicated by a grey arrow and 3F-SMN protein is indicated by a black arrow. The red square indicates the region used for quantification of SMN protein signal for Panel B and C. Panel B: SMN protein signal in cell lysates were quantified, and the fold change in SMN protein signal relative to endogenous SMN protein signal is shown. Panel C: SMN protein signal in EVs was quantified and presented as the fold change in SMN protein signal relative to endogenous SMN protein signal. Panel D: Particle size and diameter of isolated EVs was assessed by nanoparticle tracking analysis. In panels B and C, significance was calculated after transforming data by log transformation, then tested for significance using one-way ANOVA followed by Tukey’s multiple comparisons test. An asterisk (*) indicates p < 0.05, two asterisk (**) indicates p < 0.01, and three asterisk (***) indicates p < 0.001. Data represents 3 independent experiments.

Fig. 5

Application of conditioned medium containing EVs loaded with 3F-SMN protein onto A549:HA-Gemin2 cells results in FLAG-positive nuclear foci. Panel A: Conditioned medium (CM) from HepG2 cells infected with Ad3F-SMN or mock-infected was applied to A549:HA-Gemin2 cells in the presence or absence of 100 μM 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). Two h post-treatment, the cells were washed and processed for immunofluorescent staining with anti-FLAG and anti-HA, and images were captured by epifluorescence microscopy. Arrows indicate representative nuclear gem-like foci. Scale bars represent 10 µm. Panel B: FLAG-positive nuclear foci were quantified and expressed per 100 cells. Significance was calculated after transforming data by log transformation using two-way ANOVA followed by Tukey’s multiple comparisons test. Four asterisk (****) indicates p < 0.0001. Data represents 3 independent experiments.

Fig. 6

EV-delivered 3F-SMN protein is internalized by recipient A549:HA-Gemin2 cells and localizes in gem-like structures. Panel A: Confocal immunofluorescence images of A549:HA-Gemin2 cells that were treated for 2 h with mock or 3F-SMN CM, and processed with antibodies to HA and FLAG. Panel B: Images in panel A were examined through orthogonal projections generated from z-stacks. Panel C: Orthogonal projections generated of z-stacks of parental A549 cells stained with antibodies to tubulin and SMN. Scale bars represent 10 µm.

Fig. 7.

3F-SMN protein co-localizes with HA-Gemin2 in nuclear gem-like structures in A549:HA-Gemin2 cells. Panel A: Confocal immunofluorescence images of A549:HA-Gemin2 cells that were treated for 2 h with mock or 3F-SMN CM, and processed with antibodies to HA and FLAG. Panel B: The image in panel A (lower panel) was further examined through orthogonal projections generated from z-stacks. Panel C: Additional images and orthogonal projections from A549:HA-Gemin2 cells treated with 3F-SMN CM showing colocalization of 3F-SMN and HA-Gemin2 proteins. Scale bars represent 10 µm.

Fig. 8

EV-mediated delivery of mCherry-SMN and 3F-SMN protein to A549 cells and fibroblasts derived from an SMA patient. Panel A: CM from HepG2 cells infected with AdmCherry-SMN was applied to A549 cells. Two h post-treatment, the cells were washed, fixed, and stained with Hoechst to visualize nuclei. Images were captured by confocal microscopy. Panel B: CM from HepG2 cells infected with Ad3F-SMN or mock-infected was applied to fibroblasts derived from a patient with SMA. Two h post-treatment, the cells were washed and processed for immunofluorescence staining with anti-FLAG and anti-tubulin, and images were captured by confocal microscopy. Scale bars represent 10 µm.