Part:BBa_K5499010

Engineered exosome 2

To efficiently deliver the IDS protein and penetrate the blood-brain barrier, we designed a novel exosome vector based on research by João Vasco Ferreira et al.[1]. Sequence and Features

Assembly Compatibility:

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  COMPATIBLE WITH RFC[10]
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  COMPATIBLE WITH RFC[12]
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  COMPATIBLE WITH RFC[1000]

Usage and biology

According to the analysis of proteins in exosomes conducted by João Vasco Ferreira and colleagues, we added an Exosignal containing the KFERQ pentapeptide motif to the C-terminus of the IDS protein. This modification allows the IDS protein to be loaded into engineered exosomes through a LAMP2a-dependent process(Figure 1)

Figure 1: Sencond Generation Plasmid Model Diagram

Construction

First, we performed a double digestion of the pLVX-Puro vector, linearizing it with EcoR I and Xba I. After gel electrophoresis and DNA purification, we obtained the linearized plasmid vector. We then amplified the DNA fragment containing the KFERQ pentapeptide motif from an existing IDS plasmid in the lab. Using homologous recombination, we ligated the linearized vector with the target fragment. The recombinant product was transformed into DH5α competent cells for culture. After antibiotic selection, we successfully obtained positive clones, and the correctness of the construction was confirmed by PCR and sequencing (Figure 2).We constructed the fragment part into a lentiviral expression vector and then transfected it into HEK293T cells.

Figure 2: IDS-ExoSignal fragment: 1816 bp

1. Verify the morphology of the exosomes by transmission electron microscopy (TEM)

The exosomes extracted from the culture medium were characterized using transmission electron microscopy (TEM) (Figure 3) to confirm their morphology and purity.

Figure 3: Exosome Morphology Diagram by TEM

2. Verify the IDS expression in the exosomes

Western blot analysis confirmed a significant increase in IDS protein expression in the stable cell line, particularly in exosomes, where the IDS expression level was several times higher than that of the control group (Figure 4). This result demonstrates that we successfully constructed a cell line capable of high IDS expression in exosomes, meeting our design objectives.

Figure 4: Western Blot Results Diagram

3. Verify the IDS biological characteristics through different cell model

3.1 Fluorescent labeling of exosomes
We labeled the collected exosomes using DiI dye. A dye dilution was prepared, followed by incubation at room temperature in the dark. The labeled exosomes were then purified by ultrafiltration, resulting in a labeled exosome suspension.
3.2 Monolayer U87MG cell model
We conducted exosome uptake experiments on U87MG cells using flow cytometry and confocal fluorescence microscopy. The results showed that the uptake efficiency of IDS ExoSignal small extracellular vesicles (sEVs) was similar to that of unmodified IDS sEVs (Figures 5 and 6), indicating that this iteration did not significantly enhance cellular uptake of the exosomes.

Figure 5: Flow Cytometry Analysis of Exosome Uptake in Monolayer U87MG Cells

Figure 6: Confocal Fluorescence Imaging of Exosome Uptake in Monolayer U87MG Cells

3.3 In vitro Blood-Brain Barrier(BBB) model
we successfully constructed an in vitro BBB model by co-culturing human brain microvascular endothelial cells (HCMEC/D3) with astrocytoma cells (U87MG)(Figure 7).

Figure 7: In Vitro Blood-Brain Barrier Model Diagram

In the in vitro blood-brain barrier model, we found that both IDS sEVs and IDS ExoSignal sEVs exhibited low penetration efficiency and failed to effectively cross the BBB to reach U87MG cells in the lower chamber .By observing the two images (Figures 8 and 9), we can conclude that the lentiviral expression vector containing this composite part, after transfecting HEK293T cells, does not significantly enhance the engineered exosomes' ability to penetrate the blood-brain barrier.

Figure 8: Flow Cytometry Analysis of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model

Figure 9: Confocal Fluorescence Imaging of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model

===Reference=== 1. Ferreira, J.V., et al., LAMP2A regulates the loading of proteins into exosomes. Sci Adv, 2022. 8(12): p. eabm1140.