Journal: The Journal of Biological Chemistry

Article Title: Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity

doi: 10.1016/j.jbc.2021.101266

Figure Lengend Snippet: Exosome purification and characterization. A , schematic describing exosome purification procedure. B , size distribution plot of purified exosomes, as determined by NTA. C , immunofluorescence image of purified exosomes, labeled with an Alexa Fluor 647-conjugated anti-CD63 monoclonal antibody, captured using the Particle Metrix PMX-220 ZetaView camera. D , electron micrograph of negative-stained, purified exosomes. Bar, 100 nm. E , immunoblot analysis of equal proportions of 293F cell and exosome lysates using antibodies specific for the exosomal markers CD81, CD9, CD63, E-cadherin (E-cad), N-cadherin (N-cad), GRP78/BiP, calreticulin (CALR), ERGIC-3, GM130, HSP60, and HSP90. The amount and ratio of cell and exosome lysates were selected empirically to show the different enrichment of the CD81, CD9, and CD63 proteins, were kept constant in all immunoblots, by proportion equaled a 10-fold overloading of exosomes relative to cells, and by amount of protein equaled 15 μg cell lysate protein/lane and 0.15 μg exosome lysate protein/lane.

Article Snippet: Membranes were then incubated overnight in blocking solution containing antibodies obtained from commercial sources (CD9 [clone HI9a, BioLegend]; CD63 [clone MX-49.129.5, Santa Cruz Biotechnology], CD81 [555675, BD Pharmingen], E-cadherin [20874-1-AP, Thermo Fisher]; N-cadherin [MA1-2002, Thermo Fisher]; GRP78/BiP [PA1-014A, Thermo Fisher]; calreticulin [MA5-32131, Thermo Fisher]; ERGIC-3 [clone B5, sc-398778, Santa Cruz Biotechnology]; GM130 [PA1-077, Thermo Fisher]; HSP60 [ab190828, Abcam]; HSP90 [sc-13119; Santa Cruz Biotechnology]; Lamp2 [MA1-205, Thermo Fisher]).

Techniques: Purification, Immunofluorescence, Labeling, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity

doi: 10.1016/j.jbc.2021.101266

Figure Lengend Snippet: Antares2 expression in vivo following injection of Antares2 mRNA-loaded exosomes. A , combined light and bioluminescence images showing DTZ-triggered, Antares2-catalyzed light emission from the eye of mice that had been injected intravitreally with exosome/mRNA formulation and then exposed to topical DTZ solution 36 h later. Light emission, measured in radiance (photons/s/area (cm 2 )/steradian), is reflected in the color, with red denoting the areas of highest light emission and blue denoting the areas of lower but still significant light emission. Similar results were observed in a second animal that was subjected to the same treatment. Bar, 1 cm. B , combined light and bioluminescence images showing DTZ-triggered, Antares2-catalyzed light emission from the muscle of mice that had been injected i.m. in the left thigh with exosome/mRNA formulation and reinjected in both legs i.m. with DTZ solution 48 h later. This experiment was performed in a single animal. Bar, 1 cm. C , immunoblot of cell lysates prepared from HEK293 cells (HEK) and from cells that had been transfected 2 days earlier with plasmids designed to express Antares2-2a and Antares2-myc, using anti-mKate antibodies to detect the products of Antares2 expression and anti-HSP90 antibodies to confirm the presence of HEK293 proteins in each sample. D , immunoblot of tissue protein extracts collected 2 days after mice had been injected i.m. with either mRNA-loaded exosomes (animals 1 and 2) or PBS (control). Tissue protein samples were probed with ( upper blots ) antibodies specific for mKate to detect Antares2 and with ( lower blots ) antibodies specific for HSP90. Arrowheads denote position of Antares2 proteins detected by the anti-mKate antibodies in skeletal muscle of treated animals.

Article Snippet: Membranes were then incubated overnight in blocking solution containing antibodies obtained from commercial sources (CD9 [clone HI9a, BioLegend]; CD63 [clone MX-49.129.5, Santa Cruz Biotechnology], CD81 [555675, BD Pharmingen], E-cadherin [20874-1-AP, Thermo Fisher]; N-cadherin [MA1-2002, Thermo Fisher]; GRP78/BiP [PA1-014A, Thermo Fisher]; calreticulin [MA5-32131, Thermo Fisher]; ERGIC-3 [clone B5, sc-398778, Santa Cruz Biotechnology]; GM130 [PA1-077, Thermo Fisher]; HSP60 [ab190828, Abcam]; HSP90 [sc-13119; Santa Cruz Biotechnology]; Lamp2 [MA1-205, Thermo Fisher]).

Techniques: Expressing, In Vivo, Injection, Formulation, Western Blot, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity

doi: 10.1016/j.jbc.2021.101266

Figure Lengend Snippet: Exosome purification and characterization. A , schematic describing exosome purification procedure. B , size distribution plot of purified exosomes, as determined by NTA. C , immunofluorescence image of purified exosomes, labeled with an Alexa Fluor 647-conjugated anti-CD63 monoclonal antibody, captured using the Particle Metrix PMX-220 ZetaView camera. D , electron micrograph of negative-stained, purified exosomes. Bar, 100 nm. E , immunoblot analysis of equal proportions of 293F cell and exosome lysates using antibodies specific for the exosomal markers CD81, CD9, CD63, E-cadherin (E-cad), N-cadherin (N-cad), GRP78/BiP, calreticulin (CALR), ERGIC-3, GM130, HSP60, and HSP90. The amount and ratio of cell and exosome lysates were selected empirically to show the different enrichment of the CD81, CD9, and CD63 proteins, were kept constant in all immunoblots, by proportion equaled a 10-fold overloading of exosomes relative to cells, and by amount of protein equaled 15 μg cell lysate protein/lane and 0.15 μg exosome lysate protein/lane.

Article Snippet: Membranes were then incubated overnight in blocking solution containing antibodies obtained from commercial sources (CD9 [clone HI9a, BioLegend]; CD63 [clone MX-49.129.5, Santa Cruz Biotechnology], CD81 [555675, BD Pharmingen], E-cadherin [20874-1-AP, Thermo Fisher]; N-cadherin [MA1-2002, Thermo Fisher]; GRP78/BiP [PA1-014A, Thermo Fisher]; calreticulin [MA5-32131, Thermo Fisher]; ERGIC-3 [clone B5, sc-398778, Santa Cruz Biotechnology]; GM130 [PA1-077, Thermo Fisher]; HSP60 [ab190828, Abcam]; HSP90 [sc-13119; Santa Cruz Biotechnology]; Lamp2 [MA1-205, Thermo Fisher]).

Techniques: Purification, Immunofluorescence, Labeling, Staining, Western Blot