Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment

Abstract

Exosomes are cell-derived nanovesicles (50-150 nm), which mediate intercellular communication, and are candidate therapeutic agents. However, inefficiency of exosomal message transfer, such as mRNA, and lack of methods to create designer exosomes have hampered their development into therapeutic interventions. Here, we report a set of EXOsomal transfer into cells (EXOtic) devices that enable efficient, customizable production of designer exosomes in engineered mammalian cells. These genetically encoded devices in exosome producer cells enhance exosome production, specific mRNA packaging, and delivery of the mRNA into the cytosol of target cells, enabling efficient cell-to-cell communication without the need to concentrate exosomes. Further, engineered producer cells implanted in living mice could consistently deliver cargo mRNA to the brain. Therapeutic catalase mRNA delivery by designer exosomes attenuated neurotoxicity and neuroinflammation in in vitro and in vivo models of Parkinson's disease, indicating the potential usefulness of the EXOtic devices for RNA delivery-based therapeutic applications.

Fig. 1

Devices to boost exosome production. a Schematic illustration of luminescence assay for the quantification of exosome production. Ectopically expressed CD63-nluc is packaged into exosomes, which are secreted into the supernatant by the cultured cells. The ability of transgenes to increase the production of exosomes is determined by measuring the luminescent reporter in the supernatant after stepwise centrifugation to remove live cells, dead cells, and cell debris. b Effect of exosome production boosters in HEK-293T cells. CD63-nluc (pDB30: P_hCMV-CD63-nluc-pA) was expressed together with potential production boosters as follows. Control; EYFP-C1 (P_hCMV-EYFP-pA), NadB; pRK246 (P_hCMV-NadB fragment-pA) SDC4; pDB13 (P_hCMV-SDC4-pA) STEAP3; pDB12 (P_hCMV-STEAP3-pA), STEAP3:SDC4 3:2; cotransfection of pDB12 and pDB13 (ratio 3:2), STEAP3:SDC4:NadB 3:2:1; cotransfection of pDB12, pDB013, and pRK 246 (ratio 3:2:1), Bicistronic STEAP3 -SDC4; pDB59 (P_hCMV-STEAP3-IRES-SDC4-pA), bicistronic STEAP3-SDC4: Nad B 5:1; cotransfection of pDB59 and pRK246 (ratio 5:1), tricistronic STEAP3-SDC4-NadB; pDB60 (P_hCMV-STEAP3-IRES-SDC4-IRES-NadB-pA). Measured values are reported in relative luminescence units (RLU). c Result of nanoparticle tracking analysis. Concentration and size distribution of exosomes secreted from cells engineered with the exosome production booster (pDB60) were compared with those under the control (EYFP expression by EYFP-C1) conditions. Raw data is shown in Figure S2. d Exosome production booster in patient-derived mesenchymal stem cells (hMSCs). CD63-nluc (pDB30) and the exosome production booster (pDB60) were introduced into hMSCs by electroporation and CD63-nluc secreted into the supernatant was assayed. All the data are mean ± SEM of three independent experiments (n = 3). ****p < 0.0001, two-tailed Student’s t-test

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/D_box) 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/D_box: pSA462 (P_hCMV-nluc-C/D_box-pA), without C/D_box: pRK0 (P_hCMV-nluc-pA)), the exosome production booster (Pro. Booster, pDB60), cytosolic delivery helper (pDB68 (P_hCMV-Cx43 S368A-pA)), RNA packaging device (pSA465 (P_hCMV-CD63-L7Ae-pA)), and targeting module (pRVG-Lamp2b: P_hCMV-RVG-Lamp2b-pA) (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/D_box. c Assay of the effect of incorporation of C/D_box 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/D_box in its 3′-UTR (coded by pRK0, pSA462, pSA463, and pSA464, respectively; P_hCMV-CD63-L7Ae-0~5 of C/D_box(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/D_box (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

Fig. 3

Application of the EXOtic devices. a Protection against neurotoxicity in an in vitro experimental model of Parkinson’s disease by catalase mRNA delivery. b HEK-293T cells were transfected with plasmids encoding catalase mRNA with a C/D_box (pDB129: P_hCMV-Catalase-C/D_box-pA) together with the EXOtic devices (pSA465, pDB60, pDB68, and pRVG-Lamp2b) (mock: transfection with pEYFP-C1). Supernatant containing the engineered exosomes was applied to Neuro2A cells (CHRNA7-positive). Then, the cells were incubated with various 6-OHDA concentrations, and CCK-8 viability assay was conducted. c Engineered exosome producer cells were subcutaneously implanted with Matrigel in living mice (C57BL6/J) and the mice were used for further assays. d Result of nluc mRNA delivery. The exosome producer cells were transfected with pSA462 (P_hCMV-nluc-C/D_box-pA) and the EXOtic devices (with device: pSA465, pDB60, pDB68, and pRVG-Lamp2b, without device: pEYFP-C1) and the cells were subcutaneously implanted in mice. Forty-eight hours later, the brains were removed. Luminescence from whole brain homogenates was quantified. e Attenuation of neuroinflammation by catalase mRNA delivery in vivo. Exosome producer HEK-293T cells (transfected with pDB129 (P_hCMV-Catalase-C/D_box-pA), pSA465, pDB60, pDB68, and pRVG-Lamp2b. pEYFP-C1 was used as compensation for (−) condition) were subcutaneously implanted in mice. One day later, the mice were intracerebrally injected with 2.5 μg of 6-OHDA. Six days later, total RNA was extracted from the whole brain, and mRNA expression level of each marker was assayed by qPCR (GAPDH for internal standard). f Immunostaining result of TH-positive neurons. The mice were treated the same way as for e. After sacrificing the mice, midbrain sections were prepared, and immunostaining of TH-positive neurons in dorsal striatum (DStr, indicated by a dotted line) was conducted. Ruler bars represent 500 μm. g Attenuation of neuroinflammation caused by systemic LPS injection with catalase mRNA delivery in vivo. The same exosome producer cells were prepared as for e. At 2 days after implantation, 0 or 0.3 mg/kg of LPS were injected i.p.. 4 h later, RNA was extracted from the whole brain, and GFAP mRNA expression level was assayed by qPCR (GAPDH for internal standard). Error bars represent SEM (b three independent experiments (n = 3), e, f: 7–8 mice (n = 7–8).) *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed student’s t-test