Results
Transfection Experiment
Following the transfection of HEK293T cells, we evaluated the
procedure's outcome using fluorescence microscopy.
Objective: To detect eGFP expression from the transfer plasmid.
Results: The images obtained demonstrate the successful transfection
of HEK293T cells.
CONTROL
TRANSFECTED
Titration of rAAV
After the vexosome isolation procedure, we decided to conduct
qPCR to titrate the rAAV contained in exosomes.
Objective: To quantify the viral load within the vexosomes.
Results:The inconclusive data of the qPCR could not give us any
reliable insight as to the rAAV2 titer.
Treatment/Transduction experiment
We treated the testing model with vexosomes and assessed the
results by utilizing qPCR analysis and
Flow cytometry.
Objective: To detect increased expression of miRNA-195 in treated
cells via qPCR analysis and increased methylation of the
PP2A catalytic subunit via FACS analysis.
Total RNA spectrophotometry: To measure the miRNA-195 levels of the cells in the end
of our experiment, their total RNA was isolated using a kit,
and the samples’ RNA concentration and purity was measured
using Nanodrop spectrophotometry.
Results:
All of the samples had a valid RNA concentration, however,
the sample TT+x1 showed a significantly lower RNA content
than the others, possibly due to a stress reaction from the
respective cells.
qPCR Results: As shown in the graph below, a significantly higher expression level of miRNA-195 was observed in cells treated with vexosomes (TT+) compared to those treated with exosomes isolated from untransfected cells (TC+). It was also evident that the expression of miRNA-195 decreased in treated cells compared to untreated cells (T-), which was an expected outcome, considering the stress the treated cells were subjected to.
In this analysis, we were unable to detect eGFP, making it
challenging to accurately assess the methylation levels of
PP2Ac. However, we did observe some notable findings.
Specifically, Calyculin A effectively inhibited PP2A activity,
and the treated cells showed signs of stress, as anticipated.
Abbreviations Used in the Following Figures:
CA: Calyculin A treatment
T-: Untreated cells
TC: Treatment derived from control cells
TT: Treatment derived from transfected cells
X1, X2: Different dosages of therapy treatment
Ab+: Stained with methyl-PP2A-Ca/β(2Α10) antibody
After analyzing the data from qPCR and FACS, and combining this with a brief observation of our cells under fluorescence microscopy, we encountered conflicting results between the two analyses. However, further testing, thorough research, and continuous feedback from experts led us to the conclusion that transduction was successful, but the eGFP detection method failed to provide reliable results.
Proof of Concept
→ Use of iPSCs
Induced pluripotent stem cells (iPSCs) are an excellent fit for our project due to several key advantages:
- They facilitate efficient production of rAAVs following transfection.
- They allow for straightforward manipulation.
- They exhibit rapid growth in culture.
- They enhance biocompatibility and reduce the risk of immune rejection.
- They can produce significant amounts of exosomes with rejuvenating qualities to promote cell health.
These attributes collectively position iPSCs as the optimal choice for developing an innovative treatment for Alzheimer’s disease.
→ Plasmid Efficiency
Based on the successful transfection experiment and the qPCR results, we can conclude that our plasmid design is functional, leading to the expression of both miRNA-195 and eGFP. The protocol applied was also validated, as indicated by the relatively high transfection rate.
→ rAAV Production
The qPCR analysis of the load of the exosomes confirmed the successful construction of the virus within the transfected cells and validated the effectiveness of the protocols followed for both virus production and exosome isolation.
→ Modification of Exosomes
Both extensive literature data and the simulation using Agent-Based Modeling (ABM), which models the incorporation of the RVG29 peptide into the exosome membrane via Click Chemistry, are sufficient to consider this method of targeting neuronal cells strongly capable of directing our delivery vesicles directly to the brain cells of patients.
→ Vexosomes as an Innovative Delivery System
As outlined in the results above, we have compelling evidence indicating that the transduction process was successful. This leads us to the conclusion that the delivery method is reliable, effectively facilitating the expression of the miRNA-195 gene in the target cells.