Demonstration
In cerebrospinal fluid of neurodegenerative diseases’ patients, excessively high concentration of glutamate, which is an excitatory neurotransmitter, is universally detected. Glutamate accumulated in the synapse cleft and cerebrospinal fluid may keep exciting the neurons and glial cells and induce excitotoxicity to harm the cells. In order to reduce the concentration of glutamate in the cerebrospinal fluid, we used Excitatory Amino Acid Transporter 2 (EAAT2) derived from human neurons, which may absorb the cytotoxic glutamate left in extracellular matrix.
In order to ensure the effective expression of EAAT2 in astrocytes and neurons in the human brain, we used exosomes with Cx43 expressed on the cell surface, which may anchor the exosomes on human neuron and glial cells. It may also facilitate the fusion of their membranes and send mRNA enclosed to these cells.
To pack more therapeutic mRNA inside the exosomes, and avoid carrying other mRNA which may cause over-expression of unrelated exogenous genes, we designed to add the C/D Box downstream of the EAAT2-coding sequence. The C/D Box may associate with proteins such as Snu13 and L7Ae, which can be linked with membrane proteins and help to bind more mRNA on the inner surface of exosomes.
When the therapeutic EAAT2 mRNA is transfected or enclosed into exosomes, the exosomes may be injected into the human body, and deliver the mRNA to the directed neurons and glial cells.
For demonstration purposes, we used HEK293T as the exosome donor and Neuro-2a cells to mimic the astrocytes in human brain. And we conducted the followed experiments to demonstrate that our exosome medicine designed meet the listed points below:
1. The EAAT2-coding mRNA can be in vitro transcribed without premature termination.
2. Exosomes can effectively deliver the mRNA EAAT2-C/D Box into Neuro-2a cells.
3. The expression of Cx43 on exosome surface can facilitate the astrocytes’ transport of therapeutic mRNA.
4. The expression of exogenous EAAT2 facilitates the intake of glutamate and alleviate the excitotoxicity induced by excessively high level of glutamate.
Harvesting Intact mRNA
The first step of producing exosome medicine is to in vitro transcribe the therapeutic mRNA, while the IVT of mRNA longer than 500 nt may have a risk of premature termination. In our research, we found that a higher concentration of NTP, a lower temperature at about 15 Celsius and an extended reaction time to 2 hours can significantly prevent the pre-maturation of in vitro transcription.
To investigate if we could transcribe the intact EAAT2 mRNA, we used the T7 MAXIscript kit and pcDNA3.1(+) as a template to conduct a pre-experiment. After the in vitro transcription, 1 ul TURBO DNase was added and the sample was incubated at 37℃ for 15 minutes. A polyadenylation reaction was also used to protect the transcribed mRNA. A cDNA of transcribed mRNA was synthesized using the universal RT primer (before the reaction, the reagent mix was set in 85oC for 3 minutes to inactivate the DNase). An agarose gel electrophoresis was conducted to confirm the integrity of our transcribed mRNA.
Figure 1. The results of DNA agarose gel electrophoresis comparing with 1 kb ladder. The length of our amplified cDNA were approximately 3200 base pairs and 10,000 base pairs.
Demonstrated by the two separated runs of electrophoresis, this set of experiments indicates that we can produce intact transcribed mRNA by in vitro transcription.
Efficient Exosome Delivery
Our exosome medicine is designed to carry a therapeutic mRNA to the receptor cells, so successful delivery is therefore essential for this medicine to act normally as expected. To investigate if exosomes can carry the EAAT2-C/D Box transcript to the N2a, we used Cx43-transfected HEK293T to produce exosomes. After ultracentrifugation, we transfected the exosomes with the EAAT2-C/D Box transcript by using the transfecting reagent Exo-fect. The transfected exosomes were then incubated with N2a cultured in six wells in a 12-well plate with DMEM exo-free medium in each well. In other six wells, we used empty pcDNA3.1(+) as the negative control to prevent the interference of endogenous EAAT2 or EAAT2-like mRNA on the qRT-PCR results. In this experiment, a short sequence on the EAAT2 gene was used as a target and has-miR-23a-3p was used as the endogenous reference gene. The distribution of 96-well plate is listed as below:
After the qRT-PCR to detect the EAAT2-C/D Box mRNA, no signal of EAAT2 was found in the pcDNA3.1-transfected group. The ΔCt value of the EAAT2-transfected group is listed below:
Figure 2. The EAAT2 mRNA can be successfully delivered into Cx43-transfected cells while no endogenous EAAT2 or EAAT2-like mRNA was detected in N2a cells. Student’s t-test was applied with the chart (*: p<0.05, **: p<0.01, ***: p<0.001).
Brain-Targeted Delivery
To investigate if the expression of Cx43 can influence the mRNA-uptake efficiency of N2a cells, we transfected Cx43-pcDNA3.1(+) into N2a cells, we transfected Cx43-pcDNA3.1(+) in six wells of HEK293T cells, and transfected empty pcDNA3.1(+) in the rest six wells in a 12-well plate. The distribution of the 12 wells are displayed as below:
After ultracentrifugation, we transfected in vitro transcribed EAAT2-C/D Box into the harvested exosomes from each well using Exo-fect. Transfected exosomes were then incubated with N2a cultured in another 12-well plate with DMEM exo-free medium in each well. The EAAT2-coding mRNA was used as a target and has-miR-23a-3p was used as the endogenous reference gene in the following qRT-PCR. The distribution of 96-well plate is displayed below:
After the qRT-PCR, we calculated the ΔCt of each sample (Ct of target gene divided by Ct of endogenous gene), the results are listed below:
Figure 3. The ΔCt value of EAAT2 transfected in Cx43-transfected cells is significantly lower than the pcDNA3.1(+)-transfected cells, which indicates that the volume of EAAT2-coding mRNA transfected is significantly higher when the Cx43 is expressed on surface of N2a cells. Student’s t-test was applied in this investigation (*: p<0.05, **: p<0.01, ***: p<0.001).
EAAT2’s Effective Expression
EAAT2 encoded on therapeutic mRNA plays a core role in the exosome medicine for neurodegenerative diseases. To functionally characterize EAAT2, we used Neuro-2a cells derived from mouse which possess obvious and numerous branches that appear in the astrocytes in the human brain. The P3 N2a cells were cultured in a 24-well plate, all of them were transfected with EAAT2 or empty pcDNA3.1 (+). The distribution of the 24-well plate is displayed as below:
24 hours after the Engreen transfection of EAAT2 into N2a cells, we washed the cells with sterile PBS, and changed the DMEM medium to RPMI1640 phenol-free medium (with 5% exo-free FBS, 1% Penicillin and Streptomycin added). To simulate the inflammation-inducing environment in cerebrospinal fluid, we added additional glutamate to keep its concentration at approximately 80 uM. The incubation time for each sets of cells was set to be 0 hour, 3 hours, 6 hours and 9 hours, we distributed the cells as the 24-well plate displayed below:
To further investigate the influence of EAAT2 brought to N2a cells in high glutamate condition, we applied the glutamate assay and WST-1 cytotoxicity test on the treated N2a cells. When N2a cells were cultured for 0/3/6/9 hours, the media of that cells were be used and 200 ul of the remaining medium to conduct the WST-1 test. The extracted medium from each well was used to measure the glutamate concentration. And the remained 200 ul medium was added with 20 ul WST-1 solution and cultured for 2 hours, the absorbance at 450 nm (and 690 nm as a reference wavelength) was measured.
Figure 4. The increase of glutamate concentration is significantly faster in EAAT2-untransfected groups comparing to EAAT2-transfected groups, which indicates that the expression of EAAT2 can help to stabilize the concentration of glutamate in cell medium.
Figure 5. The EAAT2 may have significant impact on absorbing glutamate in the cell medium after 6 hours. Student’s t-test applied (*: p<0.05, **: p<0.01, ***: p<0.001).
Figure 6. The cytotoxicity induced by high concentration of glutamate in EAAT2-transfected cell medium can be obviously alleviated as the A450-A690 is remarkably lower than that of pcDNA3.1(+)-transfected cells.
Figure 7. After a 3-hour incubation, the effect of EAAT2 to reduce the excitotoxicity is significant as shown, Student’s t-test applied (*: p<0.05, **: p<0.01, ***: p<0.001).
