Part:BBa_K5499011

Engineer exosome 3

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and biology

Initial studies showed that unmodified exosomes, when injected intravenously, primarily accumulate in the spleen and liver, with only 0.5% reaching the brain. This finding limits the application of exosomes as a drug delivery tool for the nervous system. Therefore, to develop an effective exosome delivery system, modifications are required to improve their ability to cross the blood-brain barrier (BBB).
To address this challenge, We selected the TAT (trans-activator of transcription) peptide as a modification tool. TAT, derived from HIV-1, has strong transmembrane penetration capabilities, allowing it to quickly pass through biological membranes, including the BBB. It also has excellent endosomal and lysosomal escape abilities, making TAT-modified exosomes ideal candidates for enabling exosome penetration across the BBB. In previous studies by Zhu et al.[1], TAT-modified exosomes successfully delivered drugs to glioma cells, achieving significant tumor inhibition, providing a theoretical basis for this research.

1. Synthesis and Amplification of the TAT Gene Sequence

The TAT gene sequence (33 bp) was synthesized first. PCR was then used to amplify the TAT fragment containing homologous recombination sequences at both ends(Figure 1), in preparation for subsequent cloning.

Figure 1: TAT fragment: 106 bp

2. Amplification of Homologous Recombination Fragments

Using existing plasmids containing LAMP2A mature peptide and LAMP2A signal peptide as templates, we performed PCR to amplify fragments containing homologous recombination sequences(figure 2 and 3). The amplification was verified by agarose gel electrophoresis.

Figure 2: LAMP2A mature peptide : 1168 bp

Figure 3: LAMP2A signal peptide : 113 bp

3. Double Digestion and Purification

The pLVX-N1-ACGFP vector was digested using restriction enzymes EcoR I and Xba I. After confirming the digestion products, a DNA recovery and purification kit was used to recover the linearized plasmid fragments.

Figure 4: lineared vector plasmid : 8012 bp

4. Preparation of the Homologous Recombination Reaction

Using the ClonExpress® Ultra kit, TAT fragments, LAMP2A mature peptide, LAMP2A signal peptide, and linearized plasmid were mixed at appropriate ratios. The mixture was reacted on ice to achieve efficient recombination.

5. Transformation and Identification

The recombinant plasmids were then introduced into DH5α competent cells by electroporation. Successfully transformed cells were selected from the culture medium and identified by PCR. Finally, strains with correctly constructed plasmids, confirmed by first-generation sequencing, were selected to ensure the validity of subsequent experiments.

Figure 5: Colony PCR Result

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.

2.Verify the IDS biological characteristics through 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).
We evaluated the uptake of exosomes by U87MG cells in the lower chamber of the in vitro BBB model using flow cytometry and confocal fluorescence imaging techniques. Flow cytometry analysis （Figure 6） revealed that the uptake efficiency of exosomes in the TAT-IDS Exosignal group was 21.15%, significantly higher than that of the IDS group (3.30%) and the IDS Exosignal group (4.79%).

Figure 6: Flow Cytometry Analysis of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model

Confocal fluorescence imaging （Figure 7）further demonstrated that the red signal (indicating TAT-IDS Exosignal sEVs) in the TAT-IDS Exosignal group was markedly stronger than in the IDS and IDS Exosignal groups, as illustrated in the corresponding bar graph on the right.

Figure 7: Confocal Fluorescence Imaging of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model

In Conclusion

After BBa_K5499011 is integrated into the lentiviral expression vector, the exosomes secreted by the transfected HEK293T cells will exhibit significant properties, such as crossing the blood-brain barrier and other biological barriers. Future iGEM teams aiming to treat central nervous system diseases can consider this composite part as a drug delivery carrier

Reference

1. Zhu, Z., et al., Specific anti-glioma targeted-delivery strategy of engineered small extracellular vesicles dual-functionalised by Angiopep-2 and TAT peptides. J Extracell Vesicles, 2022. 11(8): p. e12255.