Part:BBa_K1180002:Experience

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Targeting capability validation (in vitro and in vivo)

RVG exosomes specifically deliver fluorescent-labeled oligonucleotide into neuronal cells

To determine whether RVG exosomes can deliver siRNAs into neuronal cells, Neuro2A cells were selected as the recipient cells to incubate with RVG exosomes loaded with Alexa Fluor 555-tagged oligonucleotide (red fluorescence). First, untreated Neuro2A cells or cells treated with RVG exosomes but without loading the fluorescent-labeled oligonucleotide, which served as the controls, were not fluorescently labeled under fluorescence confocal microscopy. In contrast, significant fluorescence signals were observed in Neuro2A cells treated with RVG exosomes loaded with Alexa Fluor 555-tagged oligonucleotide, whereas the fluorescence signals were dramatically lower in cells treated with unmodified exosomes loaded with Alexa Fluor 555-tagged oligonucleotide. The results suggest that RVG exosomes can specifically deliver siRNA to cells of neuronal origin, while unmodified exosomes are generally rejected by neuronal cells. Interestingly, a greater number of Alexa Fluor 555-tagged oligonucleotides accumulated in non-neuronal cells including C2C12 (skeletal muscle origin), A549 (lung origin) and MCF-7 (breast origin) when these cells were incubated with unmodified exosomes compared with those with RVG exosomes, suggesting that RVG exosomes are, in contrast, rejected by non-neuronal cells. In summary, the results indicate that RVG peptide on the exosomal membrane efficiently guides exosomes to enter neuronal cells bearing the acetylcholine receptor on their membranes but prevents exosomes from entering non-neuronal cells lacking the surface acetylcholine receptor.

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Figure 3. Confocal microscopy images of fluorescent-labeled oligonucleotide (Alexa Fluor 555, red) in untreated control cells or in cells incubated with RVG exosomes (RVG exosome), unmodified exosomes loaded with fluorescent-labeled oligonucleotide (oligonucleotide-exosome) or RVG exosomes loaded with fluorescent-labeled oligonucleotide (oligonucleotide-RVG exosome). Images of four cell lines (Neuro2A, C2C12, A549 and MCF-7) were acquired.

RVG exosomes specifically deliver MOR siRNA into neuronal cells

Subsequently, MOR siRNA levels were assayed in recipient Neuro2A cells incubated with RVG exosomes loaded with MOR siRNA. The siRNA concentrations were barely detected in untreated control cells or in cells treated with RVG exosomes or unmodified exosomes loaded with MOR siRNA. In contrast, a significant amount of siRNAs were detected in Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. As a control, C2C12 cells were treated with RVG exosomes loaded with MOR siRNA, and MOR siRNA was barely detected. Taken together, these results clearly demonstrate that the RVG peptide modification on the exosome membrane specifically guides exosomes to target neuronal cells bearing the surface acetylcholine receptor, allowing for the efficient delivery of MOR siRNA into the recipient cells.

Figure 4. Quantitative RT-PCR analysis of MOR siRNA concentrations in Neuro2A and C2C12 cells treated with RVG exosomes (RVG exosome), unmodified exosomes loaded with MOR siRNA (siRNA-exosome) or RVG exosomes loaded with MOR siRNA (siRNA-RVG exosome).

GFP levels in the brains of GFP transgenic mice decrease after tail vein injection of a plasmid that expresses GFP siRNA

To determine whether siRNA delivered via RVG exosomes can pass through the BBB and regulate endogenous gene expression, we packaged siRNA against green fluorescent protein (GFP) into RVG exosomes and injected them into GFP-transgenic mice through the tail vein. Then, the GFP levels in various tissues were determined by measuring fluorescence emission using a fluorescence microscope. Compared with control mice, injection of the RVG exosomes loaded with GFP siRNA dramatically reduced GFP levels in different parts of the brain of GFP-transgenic mice. In contrast, unmodified exosomes loaded with GFP siRNA did not induce obvious GFP silencing in mouse brain. However, while unmodified exosomes loaded with GFP siRNA had a significant effect on GFP levels in the lungs, livers and spleens of GFP-transgenic mice, RVG exosomes loaded with GFP siRNA only induced slight but non-significant GFP silencing in these tissues. The results successfully demonstrate that exosome-packaged siRNA can be delivered to various tissues, thus silencing endogenous gene expression. The results also indicate that RVG peptide on the surface of exosomes has some selectivity for neuronal tissues, which may simultaneously prevent siRNA from spreading to non-neuronal tissues.

Figure 5. Fluorescence confocal microscopy images showing sections from different tissues of GFP-transgenic mice. GFP-transgenic mice were intravenously injected with saline (control) or with GFP siRNA loaded into normal exosomes (siRNA-exosome) or RVG exosomes (siRNA-RVG exosome).

Silencing capability validation (in vitro and in vivo)

RVG exosomes loaded with MOR siRNA specifically reduce MOR expression in neuronal cells

We next evaluate the effect of RVG exosome-delivered siRNA on MOR expression in vitro. MOR expression levels were assayed in Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. Compared with control cells, MOR protein and mRNA levels were dramatically reduced by RVG exosome-delivered siRNA, while no reduction in the MOR protein and mRNA levels were observed by exosomes without the RVG peptide on their surface. The results suggest that the RVG peptide modification on the exosome membrane can specifically guides exosomes to target neuronal cells, allowing for the delivery of MOR siRNA into the neuronal cells to reduce MOR expression levels.

Figure 6. RVG exosome-delivered siRNA specifically enters Neuro2A cells and reduce MOR expression. Left panel: Western blot analysis of MOR protein levels in untreated control Neuro2A cells or cells treated with MOR siRNA loaded in normal exosomes or RVG exosomes. Right panel: qRT-PCR analysis of MOR mRNA levels in untreated control Neuro2A cells or cells treated with MOR siRNA loaded in normal exosomes or RVG exosomes.

The effects of siRNA delivered by RVG exosomes on morphine-induced CPP

MOR and its signaling pathway are known to be involved in the dependence and relapse of opioids such as morphine and heroin. Importantly, relapse always disrupts the process of opioid withdrawal. Subsequently, we focus on investigating the effect of exosomal siRNA of MOR on opioid relapse. We evaluate the consequences of MOR knockdown by exosomal siRNA in the animals by conducting the morphine-induced conditioned place preference (CPP) test, a mouse model for morphine wanting/liking behaviors. In the CPP paradigm, mice learned to associate the rewarding effect of morphine with a drug-paired environment. The CPP test was designed to mimick the process of relapse of morphine. Before conditioning, the mice showed a preference for visiting black chamber. Then, morphine dependence was developed when mice were place-conditioned by intraperitoneal injection with morphine in the white chamber on even-numbered days (on days 2, 4, 6, 8 and 10) and with saline in the black chamber on odd-numbered days (on days 3, 5, 7, 9 and 11). On day 12, CPP test 1 was conducted by allowing the mice to freely visit the morphine-paired white chamber or saline-paired black chambers. As expected, mice showed a significant preference in visiting the morphine-paired white chamber, suggesting the development of morphine dependence. Then, morphine treatment was removed for several days. On day 26, CPP test 2 was conducted and mice spent less time in the morphine-paired white chamber than the saline-paired black chamber, suggesting the disappearance of morphine dependence. Then, mice were intravenously injected with saline or with siRNAs loaded in normal exosome or RVG exosome once every two days for a total of four times, and CPP test 3 was performed on day 32. Mice maintained their natural preference for the black chamber, suggesting that MOR siRNA had no effect on the behavior of the mice. Finally, mice were relapsed on morphine on day 33, and CPP test 4 was performed the next day. Interestingly, the mice treated with RVG exosome-delivered siRNAs maintained their natural preference for the black chamber, while the mice treated with saline or with siRNAs loaded in normal exosome show preference to morphine-paired white chamber, suggesting that the MOR siRNAs delivered by RVG exosome restrain drug addiction when the mice were re-exposed to morphine.

Figure 7. The effects of siRNA delivered by RVG exosomes on morphine-induced CPP. The upper panel is represented by the value of the time mice stay in morphine-paired white chamber minus the time mice stay in saline-paired black chamber. The lower panel is the representives of the heatmap of the mouse mobility.

The effects of siRNA delivered by RVG exosomes on MOR expression in vivo

After the CPP test, mice were sacrificed, and total RNA and protein were extracted from mouse brain to evaluate the expression levels of MOR in vivo. Both MOR protein and mRNA levels were reduced in the mice treated with RVG exosome-delivered siRNA. In contrast, siRNAs delivered by unmodified exosome could not reduce MOR mRNA and protein levels in mouse brain. Thus, these results clearly demonstrate that exosomes with RVG modification passed through the BBB and delivered MOR siRNA into the central nervous system to regulate MOR expression, while natural exosomes without the RVG modification were not capable of delivering siRNA into the central nervous system or regulating target gene expression.

Figure 8. RVG exosomes can transfer MOR siRNA through the BBB and reduce MOR expression levels in vivo. Left panel: Western blot analysis of MOR protein levels in the brains of mice following injection with saline or with MOR siRNA loaded in normal exosomes or RVG exosomes. Right panel: qRT-PCR analysis of MOR mRNA levels in the brains of mice following injection with saline or with MOR siRNA loaded in normal exosomes or RVG exosomes.

User Reviews

In the project of IGEM 2015 competition, we, NJU-China, studied intensively the function of this part (BBa_K1180002) both in vitro and in vivo and expand its application by integrating it with another part that expresses MOR siRNA. We proposed a strategy of using RVG exosomes to encapsulate MOR siRNA and specifically deliver them to the brain. We also entered the experiment data in the part's page on the Registry. As a consequence, RVG exosome-delivered MOR siRNA can reversibly block or attenuate the effects of opioids, thereby functions as therapeutics for the prevention of relapse to opioid dependence following detoxification.

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