Fig. 2 | Nature Communications

Fig. 2

From: Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment

Fig. 2

EXOtic devices for mRNA delivery. a Schematic illustration of the EXOtic devices. Exosomes containing the RNA packaging device (CD63-L7Ae), targeting module (RVG-Lamp2b to target CHRNA7), cytosolic delivery helper (Cx43 S368A) and mRNA (e.g., nluc-C/Dbox) were efficiently produced from exosome producer cells by the exosome production booster. The engineered exosomes were delivered to target cells (HEK-293T cells expressing CHRNA7) and the mRNA was delivered into the target cell cytosol with the help of the cytosolic delivery helper. Finally, protein encoded in the mRNA (e.g., nluc, represented by stars) was expressed in the target cells. b Evaluation of the effect of each EXOtic device. HEK-293T cells were transfected with plasmids coding for nluc mRNA (with C/Dbox: pSA462 (PhCMV-nluc-C/Dbox-pA), without C/Dbox: pRK0 (PhCMV-nluc-pA)), the exosome production booster (Pro. Booster, pDB60), cytosolic delivery helper (pDB68 (PhCMV-Cx43 S368A-pA)), RNA packaging device (pSA465 (PhCMV-CD63-L7Ae-pA)), and targeting module (pRVG-Lamp2b: PhCMV-RVG-Lamp2b-pA26) (for (–) condition, pEYFP-C1 was used as compensation). Cell culture supernatant containing engineered exosomes was applied to target cells. The target cell pellet was assayed for nluc activity at 24 h after supernatant transfer. The asterisk indicates nluc mRNA without C/Dbox. c Assay of the effect of incorporation of C/Dbox in the mRNA 3’UTR. HEK-293T cells were transfected with a plasmid coding for nluc bearing 0, 1, 3, or 5 repeats of C/Dbox in its 3′-UTR (coded by pRK0, pSA462, pSA463, and pSA464, respectively; PhCMV-CD63-L7Ae-0~5 of C/Dbox(es)-pA). The cell culture supernatant containing engineered exosomes was applied to target cells and nluc expression was assayed at 24 h after the medium transfer. d Evaluation of the whole EXOtic devices in patient-derived hMSCs. Patient-derived hMSCs were transfected with the plasmid coding for nluc-C/Dbox (pSA462) as well as the EXOtic devices (pSA465, pDB60, pDB68, and pRVG-Lamp2b) or pEYFP-C1 (without device) by electroporation. The cell culture supernatant containing engineered exosomes was applied to target cells and nluc expression was assayed at 24 h after medium application (Note that Y-axis of c and d is not comparable because of several factors including different sender/receiver ratio). Error bars represent SEM of three independent experiments (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-tailed Student’s t-test

Back to article page