Development of human targeted extracellular vesicles loaded with shRNA minicircles to prevent parkinsonian pathology

Abstract

Background: Neurological disorders are the second leading cause of death and the leading cause of disability in the world. Thus, the development of novel disease-modifying strategies is clearly warranted. We have previously developed a therapeutic approach using mouse targeted rabies virus glycoprotein (RVG) extracellular vesicles (EVs) to deliver minicircles (MCs) expressing shRNA (shRNA-MCs) to induce long-term α-synuclein down-regulation. Although the previous therapy successfully reduced the pathology, the clinical translation was extremely unlikely since they were mouse extracellular vesicles.

Methods: To overcome this limitation, we developed a source of human RVG-EVs compatible with a personalized therapy using immature dendritic cells. Human peripheral blood monocytes were differentiated in vitro into immature dendritic cells, which were transfected to express the RVG peptide. RVG-EVs containing shRNA-MCs, loaded by electroporation, were injected intravenously in the α-synuclein performed fibril (PFF) mouse model. Level of α-synuclein, phosphorylated α-synuclein aggregates, dopaminergic neurons and motor function were evaluated 90 days after the treatment. To confirm that EVs derived from patients were suitable as a vehicle, proteomic analysis of EVs derived from control, initial and advanced Parkinson's disease was performed.

Results: The shRNA-MCs could be successfully loaded into human RVG-EVs and downregulate α-synuclein in SH-SY5Y cells. Intravenous injection of the shRNA-MC-loaded RVG-EVs induced long-term downregulation of α-synuclein mRNA expression and protein level, decreased α-synuclein aggregates, prevented dopaminergic cell death and ameliorated motor impairment in the α-synuclein PFF mouse model. Moreover, we confirmed that the EVs from PD patients are suitable as a personalized therapeutic vehicle.

Conclusion: Our study confirmed the therapeutic potential of shRNA-MCs delivered by human RVG-EVs for long-term treatment of neurodegenerative diseases. These results pave the way for clinical use of this approach.

Keywords: Gene therapy; Human targeted extracellular vesicles; Parkinson’s disease; ShRNA minicircles; α-Synuclein.

Fig. 1

Optimization of loading and validation of h-RVG-EVs loaded with anti-α-synuclein shRNA-MCs. a, b Nanoparticle tracking analysis characterization of EVs isolated from control hDCs (a) and hDCs electroporated with hLamp2b-RVG mRNA (b). c Western blot analysis to assess expression of markers in control EV and RVG-EV. d Representative scanning electron microscopy images of EV isolated from control hDCs and hDCs electroporated with hLamp2b-RVG mRNA. Scale bar, 200 nm. e, f SH-SY5Y cells overexpressing α-synuclein were treated with anti α-synuclein shRNA-MC using a transfection reaction (TR), or loaded into control h-EVs (EV) and h-RVG-EV (RVG-EV). α-synuclein mRNA (e) and protein (f) levels were quantified and normalized to control cells. Typical western blots are shown. Data are expressed as mean ± SEM (n = 4). *P < 0.05, non-parametric Kruskal–Wallis test, statistical analyses compared to untreated control cells

Fig. 2

α-synuclein downregulation in several brain regions of mice treated with syn-MCs delivered by h-RVG-EVs. Analyses of α-synuclein mRNA expression (a, c, e) and protein (b, d, f) levels normalized to actin in the ipsilateral midbrain (a, b), striatum (c, d) and cortex (e, f) of α-synuclein PFF-treated mice (PFF) after treatment with h-RVG-EVs loaded with anti-α-synuclein (H-Syn) or GFP (H-GFP) shRNA-MCs. A group of mice was treated with anti-α-synuclein shRNA-MCs delivered by Ms-RVG-EV (Ms-Syn). Typical western blots are shown. Data are expressed as mean ± SEM (n = 6–8). *P < 0.05 compared to control mice, one-way ANOVA

Fig. 3

Effect of h-RVG-EVs loaded with syn-MCs on α-synuclein pathology in SNc. a Immunofluorescent images of midbrain sections stained with antibodies to S129 phospho-α-synuclein (green) and TH (red). Scale bar, 50 μm. b Magnified images of stained midbrain section showing co-localization of α-synuclein aggregates (green) in dopaminergic neurons (red). Scale bar, 10 μm. c Quantitation of α-synuclein-positive aggregates per section in the ipsilateral SNc of α-synuclein PFF-treated mice following treatment with h-RVG-EVs loaded with anti-α-synuclein (H-Syn) or GFP (H-GFP) shRNA-MCs or after treatment with Ms-RVG-EVs loaded with anti-α-synuclein shRNA-MCs (Ms-Syn). Data are expressed as mean ± SEM (n = 6–10). *P < 0.05 compared to PFF mice, one-way ANOVA

Fig. 4

α-Synuclein pathology in the striatum, cortex and amygdala after treatment with h-RVG-EVs loaded with syn-MCs. Quantification of aggregates in the ipsilateral striatum (a), cortex (b) and amygdala (c) of α-synuclein PFF-injected mice (PFF) following treatment with h-RVG-EVs loaded with anti-α-synuclein (H-Syn) or GFP (H-GFP) shRNA-MCs or after treatment with anti-α-synuclein shRNA-MCs loaded in Ms-RVG-EVs (Ms-Syn). Representative immunohistochemical images of intraneuronal phospho-α-synuclein-positive aggregates in the ipsilateral striatum, cortex and amygdala are shown. Scale bar, 100 µm. Data are expressed as mean ± SEM (n = 6–10), *P < 0.05, one-way ANOVA compared to PFF mice

Fig. 5

Syn-MC h-RVG-EV therapy prevented dopaminergic dysfunction and motor impairments. a TH staining of dopaminergic neurons in coronal midbrain sections of α-synuclein PFF-injected mice (PFF) following treatment with h-RVG-EVs loaded with anti-α-synuclein (H-Syn) or GFP (H-GFP) shRNA-MCs or after treatment with anti-α-synuclein shRNA-MCs loaded in Ms-RVG-EVs (Ms-Syn). Arrows point to area of decreased dopaminergic neurons. Numbers of nigral dopaminergic neurons were quantified by unbiased stereology on each brain hemisphere (I, ipsilateral; C, contralateral). b TH quantitation by optical density in ipsilateral sections of anterior, medial and posterior striatum normalized to contralateral striatum. Representative striatal section showing TH staining are shown. c Quantitative analysis of the hind limb clasping scores. d Wire hanging performance. e Time to turn around and move up toward the top of the platform in the negative geotaxis test. If the mouse was unable to turn, the default value of 30 s was taken as maximal severity of impairment. Data are expressed as mean ± SEM (n = 6–10). *P < 0.05, ***P < 0.001 compared to control mice, one-way ANOVA

Fig. 6

Effect on α-synuclein mRNA and protein levels in the spinal cord, intestine and olfactory bulb. Quantitation of α-synuclein mRNA (a, c, e) and protein (b, d, f) levels in the olfactory bulb (a and b), spinal cord (c and d) and intestine (e and f) from α-synuclein PFF-treated mice (PFF) at 90 days following treatment with h-RVG-EVs loaded with anti-α-synuclein (H-Syn) or GFP (H-GFP) shRNA-MCs or after treatment with anti-α-synuclein shRNA-MCs loaded in Ms-RVG-EVs (Ms-Syn). Typical western blots are shown. Data are expressed as mean ± SEM (n = 6–8). *P < 0.05 compared to control mice, one-way ANOVA

Fig. 7

Proteomic analysis of EVs produced by DCs isolated from control, initial PD and advance PD. a, b Heat map analysis showing differentially expressed proteins between initial PD (a) or advanced PD (b) and control patients. c Venn diagram of differentially expressed proteins detected in EVs derived from initial and advanced PD