Content

Introduction

We aim to develop engineered exosomes capable of crossing the blood-brain barrier (BBB) to alleviate central nervous system damage in patients with MPS II. After multiple trials, we designed three generations of plasmids.

The first-generation recombinant plasmid successfully integrated the IDS gene into the genome of receptor cells, allowing for high IDS protein expression, though the IDS content in exosomes was minimal.

In the second-generation recombinant plasmid, ExoSignal was added after IDS, which increased IDS sorting into exosomes, but the exosomes still could not cross the in vitro BBB model.

Finally, in the third-generation plasmid, we integrated the cell-penetrating peptide TAT alongside the IDS gene into the genome, ultimately producing engineered exosomes capable of crossing the BBB while carrying the IDS enzyme. This provides a novel approach and technical avenue for treating MPS II.

CYCLE1: Integration of the IDS Gene into the Receptor Cell Genome

Design

Our goal was to construct a stable cell line expressing the Iduronate-2-sulfatase (IDS) gene, which could then be used to extract IDS-containing exosomes for further cell and animal experiments. The IDS gene (NCBI ID: 3423) is located on chromosome Xq28 and encodes a lysosomal enzyme that degrades glycosaminoglycans (GAGs), with a protein size of 61.8 kDa.

To achieve long-term stable IDS expression, we chose the commonly used lentiviral vector pLVX-Puro, which contains a CMV promoter and a puromycin resistance gene, allowing stable integration of foreign genes into the host genome. Our design involved cloning the IDS gene into the pLVX-Puro vector (Figure 1) and achieving stable expression in HEK293T cells via lentivirus-mediated gene transfection, followed by exosome extraction to obtain IDS-containing exosomes.

Figure 1. First Generation Plasmid Model Diagram

Build

Plasmid construction: The pLVX-Puro vector was linearized by double digestion with the restriction enzymes EcoR I and Xba I. We then successfully inserted the IDS gene into the pLVX-Puro vector using homologous recombination technology. The recombinant plasmid was validated by PCR and Sanger sequencing, and positive clones were expanded for bacterial culture, preserving the successfully constructed plasmid.

Construction of stable cell lines: When HEK293T cells reached an appropriate density, we used the three-plasmid system (pMD2.G, psPAX2, and pLVX-IDS) for lentiviral packaging. The packaged viral supernatant was then used to transfect HEK293T cells, and puromycin selection was applied to obtain a stable IDS-expressing cell line.

Exosome extraction: To obtain IDS-containing exosomes, we employed differential centrifugation to extract exosomes from the stable cell line. Gradual centrifugation at different speeds was performed to remove cellular debris and larger particles, with purified exosomes ultimately obtained through ultracentrifugation (Figure 2). The extracted exosomes were characterized using transmission electron microscopy (TEM) (Figure 3) to confirm their morphology and purity.

Figure 2. Centrifugation Steps Diagram

Figure 3. Exosome Morphology Diagram

Result

Through plasmid construction and lentivirus-mediated gene transfection, we successfully inserted the IDS gene into the pLVX-Puro vector and established a stable IDS-expressing HEK293T cell line. Western blot analysis showed a significant increase in IDS protein expression in the stable cell line, confirming the success of our plasmid construction and cell line selection. However, when we analyzed the extracted exosomes, the IDS content was extremely low and barely detectable, despite the high intracellular IDS expression levels (Figure 4). This indicates that the exosomal sorting efficiency of IDS in our stable expression system was low.

Figure 4. Western Blot Results Diagram

Learn

Upon detailed analysis and reflection, we realized that simply increasing the expression level of the IDS gene within cells does not effectively enhance IDS sorting into exosomes. As a lysosomal enzyme, IDS likely requires specific signals or mechanisms for efficient sorting into exosomes. Thus, our experiment demonstrated that overexpression mediated by lentiviral vectors alone is insufficient to meet our goal. In future experiments, we plan to improve the recombinant plasmid by potentially adding specific signal peptide sequences to enhance IDS sorting into exosomes. These experimental results provided us with valuable insights, advancing our understanding of exosome biology and protein sorting mechanisms, while offering clear directions for future research.

CYCLE2: Adding the Exosignal peptide to the C-terminus of IDS to enhance its content in exosomes

Design

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]. We added the KFERQ pentapeptide motif to the C-terminus of the IDS protein, which is known to enhance exosome loading and release efficiency via a LAMP2a-mediated pathway (Figure 5).

Figure 5. Second Generation Plasmid Model Diagram

Build

1.Plasmid 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 6).

Figure 6. IDS-ExoSignal fragment: 1816 bp

2.Construction of Stable Cell Lines

To establish a stable IDS-ExoSignal cell line, we used a three-plasmid system for lentiviral packaging in HEK293T cells. When the cell density reached an optimal level, we transfected the cells with virus supernatant containing IDS-ExoSignal using PEI. After transfection, the medium was replaced with fresh complete culture medium, and successfully transfected cells were selected. Ultimately, we obtained a batch of stable IDS-ExoSignal expressing cell lines, which were preserved through long-term culture and cryopreservation techniques, ensuring a reliable source of cells for subsequent experiments.

3.Exosome Extraction

We used differential centrifugation to extract exosomes from the conditioned medium of the stable cell lines. Stepwise centrifugation at different speeds was performed to remove cell debris and larger particles, with purified exosomes eventually obtained through ultracentrifugation (Figure 7). Transmission electron microscopy (TEM) was used to characterize the morphology and purity of the extracted exosomes (Figure 8).

Figure 7. Centrifugation Steps Diagram

Figure 8. Exosome Morphology Diagram

4.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 (Figure 9).

Figure 9. Fluorescently Labeled Exosome Diagram

5.In Vitro Blood-Brain Barrier Model Construction

Through discussions with Dr. Liu Yuesheng, we learned that current enzyme replacement therapies are limited by the blood-brain barrier (BBB), preventing effective entry into the central nervous system. To address this, we successfully constructed an in vitro BBB model by co-culturing human brain microvascular endothelial cells (HCMEC/D3) with astrocytoma cells (U87MG) (Figure 10), which was used to assess the efficiency of second-generation exosomes in penetrating the in vitro BBB model. The integrity and permeability of the model were validated using an RB-Dextran leakage assay (Figure 11), laying the groundwork for future studies on exosome permeability.

Figure 10. In Vitro Blood-Brain Barrier Model Diagram

Figure 11. Diagram of RB-Dextran Solution Leakage Detection in an In Vitro BBB Model

Result

1.IDS Protein Expression:

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 12). This result demonstrates that we successfully constructed a cell line capable of high IDS expression in exosomes, meeting our design objectives.

Figure 12. Western Blot Results Diagram

2.Exosome Uptake:

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 13 and 14), indicating that this iteration did not significantly enhance cellular uptake of the exosomes.

Figure 13. Flow Cytometry Analysis of Exosome Uptake in Monolayer U87MG Cells

Figure 14. Confocal Fluorescence Imaging of Exosome Uptake in Monolayer U87MG Cells

3.BBB Penetration Experiment:

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 (Figures 15 and 16). Although exosomes performed well in cell-to-cell communication, challenges remain in overcoming the BBB. This presents new directions for future research, where our focus will be on optimizing surface modifications of exosomes to improve their BBB penetration ability and enhance the therapeutic potential of IDS protein in the nervous system.

Figure 15. Flow Cytometry Analysis of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model

Figure 16. Confocal Fluorescence Imaging of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model

Learn

This study successfully established a stable cell line expressing IDS-enriched exosomes and explored both the exosome uptake efficiency in cells and their ability to permeate the blood-brain barrier (BBB). Although we achieved certain successes in our design, we still face challenges in enabling exosomes to cross the BBB. Future research will focus on exploring new modification strategies to enhance the permeability of exosomes through the BBB.

CYCLE 3: Construction and Validation of a TAT-Modified Exosome System for Drug Delivery Across the Blood-Brain Barrier

Design

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, researchers have 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.[2], TAT-modified exosomes successfully delivered drugs to glioma cells, achieving significant tumor inhibition, providing a theoretical basis for this research.

In this context, CYCLE 3 designed an engineered TAT-IDS fusion protein, linking the TAT peptide to the N-terminal signal peptide of the Lamp2a protein to ensure its expression on the exosome membrane surface. Additionally, a P2A cleavage peptide was used to achieve co-expression of Lamp2a and IDS (iduronate-2-sulfatase) proteins, ensuring the functional expression of both proteins on the same vector (Figure 17).

Figure 17. First Generation Plasmid Model Diagram

Build

1.Plasmid Construction

·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, in preparation for subsequent cloning.

·Amplification of Homologous Recombination Fragments: Using existing plasmids containing Lamp2a and IDS as templates, we performed PCR to amplify fragments containing homologous recombination sequences. The amplification was verified by agarose gel electrophoresis.

·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.

·Preparation of the Homologous Recombination Reaction: Using the ClonExpress® Ultra kit, TAT fragments, Lamp2a fragments, IDS fragments, and linearized plasmid were mixed at appropriate ratios. The mixture was reacted on ice to achieve efficient recombination.

2.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.

3.Lentivirus Packaging and Transfection

During the construction of a stable cell line, the lentivirus system was first transfected into HEK293T cells. When cell density reached 70%, transfection was performed. After 24 hours, the medium was replaced with a complete growth medium and cultured for an additional 48 hours. Viral supernatants were collected for viral extraction. Puromycin selection was used to screen transfected cells, ultimately resulting in a stable cell line expressing the TAT-IDS fusion protein, which provides a reliable model for further experiments.

4.Exosome Extraction

We used differential centrifugation to extract exosomes from the conditioned media of stable cell lines. The media underwent stepwise centrifugation at different speeds to remove cellular debris and larger particles. Finally, purified exosomes were obtained via ultracentrifugation (Figure 18). The extracted exosomes were characterized using transmission electron microscopy (TEM) to confirm their morphology and purity (Figure 19).

Figure 19. Centrifugation Steps Diagram

Figure 19. Exosome Morphology Diagram

Fluorescent Labeling of Exosomes We labeled the collected exosomes using DiI dye. A dye dilution solution was first prepared, followed by incubation in the dark at room temperature. The labeled exosome suspension was then obtained through ultrafiltration (Figure 20).

Figure 20. Fluorescently Labeled Exosome Diagram

In Vitro Blood-Brain Barrier Model Construction We successfully constructed an in vitro blood-brain barrier (BBB) model by co-culturing human brain microvascular endothelial cells (HCMEC/D3) with astrocytoma cells (U87MG) (Figure 21). This model was used to assess the efficiency of the third-generation exosomes in penetrating the BBB. The RB-Dextran leakage assay was used to verify the integrity and permeability of the model (Figure 22), laying the foundation for studying exosome penetration efficiency in future experiments.

Figure 21. In Vitro Blood-Brain Barrier Model Diagram

Figure 22. Diagram of RB-Dextran Solution Leakage Detection in an In Vitro BBB Model

Result

1.Validation of Recombinant Plasmid Agarose gel electrophoresis was used to verify the synthesized TAT gene sequence.

Table 1. TAT Gene Sequence

Figure 23. TAT fragment - 106 bp

Figure 24. Fusion of LAMP2A Signal Peptides and TAT Fragment Results

Figure 25. LAMP2A Mature Fragment - 1168 bp

2.IDS Protein Expression Detection

Western blot analysis revealed that the expression level of IDS protein in the TAT-IDS Exosignal cell line was significantly increased compared to the HEK293T control cell line. Additionally, the IDS protein content in exosomes was notably elevated (Figure 27). These results indicate that the TAT modification did not impair the drug-loading capacity of the exosomes, supporting their potential value in drug delivery applications.

Figure 26. Western Blot Results Diagram

3.Exosome Penetration in the Blood-Brain Barrier Model

The exosome uptake in the in vitro blood-brain barrier model was assessed using flow cytometry and confocal fluorescence microscopy. The experimental results demonstrated that TAT-IDS Exosignal exosomes exhibited significantly higher penetration capabilities compared to IDS and IDS Exosignal exosomes (Figures 28, 29). This suggests that TAT modification can effectively enhance the ability of exosomes to penetrate the blood-brain barrier, providing a novel approach to drug delivery.

Figure 27. Flow Cytometry Analysis of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model

Figure 28. Confocal Fluorescence Imaging of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model

Learn

This study confirmed the feasibility of using TAT-modified exosomes for drug delivery and blood-brain barrier penetration, suggesting that this strategy could offer new technical methods and approaches for treating central nervous system diseases like MPS II. By combining flow cytometry and confocal fluorescence imaging, the effectiveness of TAT-modified exosomes was further validated, laying a solid foundation for future clinical applications. This study offers a novel approach and technical tool for the treatment of MPS II, revealing the immense potential of engineered exosomes as drug carriers.