iGEM 2024 — Team nwu-china-a

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

{% extends "layout.html" %} {% block page\_content %}

## Engineering Success

[ ]

# Content

[ ]

[Introduction](#anchor1)

[ ]

[CYCLE1](#anchor2)

[ ]

[CYCLE2](#anchor3)

[ ]

[CYCLE3](#anchor4)

[ ]

[References](#anchor5)

## 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.

![](https://static.igem.wiki/teams/5499/engineering/1.png)

**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.

![](https://static.igem.wiki/teams/5499/engineering/2.png)

**Figure 2. Centrifugation Steps Diagram**

![](https://static.igem.wiki/teams/5499/engineering/3.png)

**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.

![](https://static.igem.wiki/teams/5499/engineering/4.png)
**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).

![](https://static.igem.wiki/teams/5499/engineering/5.png)

**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).

![](https://static.igem.wiki/teams/5499/engineering/6.png)

**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).

![](https://static.igem.wiki/teams/5499/engineering/7.png)

**Figure 7. Centrifugation Steps Diagram**

![](https://static.igem.wiki/teams/5499/engineering/8.png)

**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).

![](https://static.igem.wiki/teams/5499/engineering/20.png)

**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.

![](https://static.igem.wiki/teams/5499/engineering/10.png)

**Figure 10. In Vitro Blood-Brain Barrier Model Diagram**

![](https://static.igem.wiki/teams/5499/engineering/11.png)

**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.

![](https://static.igem.wiki/teams/5499/engineering/12.png)
**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.

![](https://static.igem.wiki/teams/5499/engineering/13-pic.jpg)
**Figure 13. Flow Cytometry Analysis of Exosome Uptake in Monolayer U87MG Cells**

![](https://static.igem.wiki/teams/5499/engineering/14.png)
**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.

![](https://static.igem.wiki/teams/5499/engineering/15-pic.jpg)
**Figure 15. Flow Cytometry Analysis of Exosome Uptake in U87MG Cells in the Lower Chamber of
an In Vitro BBB Model**

![](https://static.igem.wiki/teams/5499/engineering/16-pic.jpg)
**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).

![](https://static.igem.wiki/teams/5499/engineering/17.png)

**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).

![](https://static.igem.wiki/teams/5499/engineering/18.png)

**Figure 19. Centrifugation Steps Diagram**

![](https://static.igem.wiki/teams/5499/engineering/19.png)

**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).

![](https://static.igem.wiki/teams/5499/engineering/20.png)

**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.

![](https://static.igem.wiki/teams/5499/engineering/21.png)

**Figure 21. In Vitro Blood-Brain Barrier Model Diagram**

![](https://static.igem.wiki/teams/5499/engineering/22.png)

**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.

![](https://static.igem.wiki/teams/5499/engineering/table.png)
**Table 1. TAT Gene Sequence**

![](https://static.igem.wiki/teams/5499/engineering/23.png)
**Figure 23. TAT fragment - 106 bp**

![](https://static.igem.wiki/teams/5499/engineering/24.png)
**Figure 24. Fusion of LAMP2A Signal Peptides and TAT Fragment Results**

![](https://static.igem.wiki/teams/5499/engineering/25.png)
**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.

![](https://static.igem.wiki/teams/5499/engineering/26.png)
**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.

![](https://static.igem.wiki/teams/5499/engineering/engineering-27-new.png)
**Figure 27. Flow Cytometry Analysis of Exosome Uptake in U87MG
Cells in the Lower Chamber of an In Vitro BBB Model**

![](https://static.igem.wiki/teams/5499/engineering/28-pic.jpg)
**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.

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

     [2] 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.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %} {% block page\_content %}

## Results

[ ]

# Menu

[ ]

[Introduction](#anchor1)

[ ]

[1. Plasmid Construction](#anchor2)

[1.1 Design and Amplification of the TAT Gene Sequence](#anchor2.1)

[1.2 Gene Amplification](#anchor2.2)

[1.3 Linearization of the Plasmid Vector](#anchor2.3)

[1.4 Homologous Recombination Reaction](#anchor2.4)

[1.5 Transformation and Identification](#anchor2.5)

[ ]

[2. Construction of a Stably Transfected Cell
Line](#anchor3)

[2.1 Lentivirus Packaging](#anchor3.1)

[2.2 Cell Transfection and Selection](#anchor3.2)

[ ]

[3.Expression Verification](#anchor4)

[3.1 Exosome Extraction and Morphological Verification](#anchor4.1)

[3.2 Western Blot Analysis](#anchor4.2)

[ ]

[4.Validation in the Blood-Brain Barrier Model](#anchor5)

[4.1Fluorescent Labeling of Exosomes](#anchor5.1)

[4.2 Construction of In Vitro Blood-Brain Barrier Model](#anchor5.2)

[4.3. Detection of the In Vitro Blood-Brain Barrier](#anchor5.3)

[4.4 Flow Cytometry Analysis](#anchor5.4)

[4.5. Confocal Fluorescence Imaging](#anchor5.5)

[ ]

[Summary](#anchor6)

## Introduction

This year, NWU-CHINA-A team aims to develop engineered exosomes capable of carrying IDS
and
crossing the blood-brain barrier (BBB) to alleviate central nervous system damage in
patients
with MPS II. We plan to design a lentiviral expression plasmid, which, after stable
transfection
into HEK293T cells, will enable the stable expression of the engineered exosomes
described
above. A series of experiment will be conducted to verify the biological characteristics
of
these exosomes.

## 1. Plasmid Construction

## 2. Cell Transfection

### 2.1 Lentivirus Packaging

In this experiment, we first constructed the pLVX-TAT-IDS plasmid and combined it with
pMD2.G and psPAX2 plasmids to form a three-plasmid system. Next, we used
polyethyleneimine (PEI) as the transfection reagent to transfect these plasmids into
HEK293T cells to produce the lentiviral vector. After transfection, we replaced the
culture medium 24 hours later to remove unbound plasmids and transfection reagents.
After 48 hours of incubation, we collected the cell culture supernatant and removed cell
debris through centrifugation and filtration, ultimately obtaining a high-purity viral
solution.

#### **Table3 Three-Plasmid Lentivirus Packaging System**

| Reagnet | Reaction System |
| --- | --- |
| pMD2.G | 1.25 μg |
| psPAX2 | 3.75 μg |
| pLVX-TAT-IDS Exosignal | 5 μg |
| PEI | 30 μL |
| opti-MEM | 500μL |

### 2.2 Cell Transfection and Selection

During cell transfection, we seeded HEK293T cells in a 6-well plate and added the
prepared viral solution when the cell density reached approximately 40%. After 48 hours
of incubation, the viral infection rate within the cells significantly increased. We
then added culture medium containing 3 µg/ml puromycin for selection, aiming to
gradually eliminate cells that were not successfully transfected. After several rounds
of selection, we ultimately obtained a stable cell line expressing TAT-IDS Exosignal.

## 3.Expression Verification

## 4.Validation in the Blood-Brain Barrier Model

## 5.Summary

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