Once our concept was established, we focused on designing the necessary laboratory tools. To ensure the successful completion of the development, we adopted the leading engineering methodology, Design-Build-Test-Learn (DBTL). This approach allowed us to maintain control of the process at every step.
Starting with the design phase, deeply rooted in continuous interaction with medical professionals, laboratory experts, and patients, we ensured our strategy was tailored to the challenges we encountered during our research. We continued by breaking down our experiments into specific tasks, with each one of them following the DBTL engineering cycle, allowing us to identify both strengths and weaknesses, ultimately enhancing the innovation and efficiency of our outcomes.
Cycle 1: Transfecting HEK293T
Design
HEK293T cell culture was transfected with the three rAAV-assembling plasmids. The transfer plasmid, designed by the Wet Lab team, carries the DNA of the vector, including the miRNA-195 gene and the eGFP gene. The helper plasmid contains the genome necessary for the expression of proteins, responsible for virus replication. The packaging plasmid (RepCap) encodes both the replication gene and the capsid gene required for viral genome replication and packaging, and the formation of the vector's capsid.
Build
The transfection experiment was conducted under the appropriate protocol, detailed in our lab notebook. The three plasmids used were the transfer (transgene) plasmid, the pHelper, and the RepCap, as mentioned above.
Test
The process was evaluated using fluorescence microscopy. We confirmed the transfection was successful by detecting the expression of eGFP.
Learn
The success of the procedure led us to the conclusion that both the plasmid design and the protocol development and execution were effective.
Cycle 2: Exosome Isolation
Design
Exosome isolation methods vary regarding cost, complexity, accuracy, and yield. After having assessed approaches like ultracentrifugation and immunoaffinity, we concluded that chemical precipitation using the Exo-Prep reagent was the method that best suited our experimental requirements, due to its high specificity and purity.
Build
The isolation of exosomes was carried out according to the protocol provided by the Exo-Prep supplying company.
Test
Based on the efficiency of our isolation method, we expected to observe a distinct pellet of exosomes after the experiment. While such a pellet was indeed present, we wanted to further characterize and quantify the isolated exosomes, leading us to choose Transmission Electron Microscopy (TEM). However, technical issues ultimately prevent it from conducting the analysis. As a result, for the evaluation of our design, we relied on the high yield of chemical precipitation and the subsequent DNA precipitation of the sample, both of which strongly indicate exosome presence.
Learn
The high yield of exosomes provided us with the indication that our method works. However, since our testing was not sufficient, we explored a range of analyses like Western Blot or Flow Cytometry to identify exosomes based on their specific markers, such as CD63 or CD9, for future experiments.
Cycle 3: Quantification of vexosome load
Design
Upon completing the vexosome isolation process, we decided to quantify the amount of rAAV encapsulated within the exosomes. Assessing the viral load present could provide us with critical data regarding the optimal dosage for the transduction process and the overall success of the vexosome production.
Build
To achieve our objective, we chose to perform qPCR as a method to accurately quantify the amount of vector loaded.
Test
We performed qPCR analysis on the contents of the exosomes by disrupting their membranes in accordance with a specific protocol. The viral DNA was split from capsids and was isolated, precipitated and finally eluted to prepare samples ready for qPCR. As a reference gene, we prepared a known concentration elution of our TransferGene plasmid. The results of the analysis using the DeltaDeltaCt method were unfortunately inconclusive and did not provide any significant results.
Learn
The inconclusive data of the qPCR, although inicating the prescence of viral DNA, were not reliable and could not give us any insight as to the rAAV2 titer. In the future, we plan on using triplicates for each sample, to aid us in data analysis and acquisition of more accurate results.
Cycle 4: Developing Testing Model
Design
Since our HEK293T cell culture shows normal levels of methylation of the PP2A catalytic subunit, we employed Calyculin A to achieve the inhibition of PP2A activity and mimic the pathology of an Alzheimer's brain.
Build
We treated our cell culture with a concentration of 10nM and incubated for 1h.
Test
As a result, cells appeared to be quite stressed and started to lose their adherent ability.
Learn
We concluded that cell death occurred due to the high Calyculin A dosage over an extended period of time.
Rebuild
We proceeded with the Calyculin A treatment for the second time, by lowering the dosage to 2nM and the exposure time to 30min.
Test
We observed our cells again regarding their viability and morphology, under the microscope.
Learn
We reached a conclusion that the concentration used in the second treatment was the optimal for inducing the appropriate level of cell stress without causing cell death, achieving the desirable outcome, as it was later on proved by Flow Cytometry technique.
Cycle 5: Treatment/Transduction
Design
HEK293T cells were treated with isolated vexosomes, containing rAAVs, with the aim of achieving transduction and subsequent expression of miRNA-195.
Build
To ensure the effective uptake of vexosomes by the cultured cells, we tested different dosages of treatment to determine the optimal amount that would result in successful transduction.
Test
Our experiment was conducted twice in order to obtain cells for PCR analysis, Fluorescence Microscopy, and Flow Cytometry. PCR was employed to quantify the expression of miRNA-195 in the treated cells and compare it with control cells treated with exosomes isolated from untransfected cells. Fluorescence Microscopy was the quickest way to evaluate the success of transduction. Flow cytometry allowed us to detect eGFP, expressed by transduction cells, and variations in PP2Ac methylation before and after treatment. While the results of PCR analysis were promising about the transduction showing an increase of miRNA-195 expression, in Flow Cytometry and Fluorescence Microscopy we failed to detect the expression of eGFP.
Learn
Challenged by these results we embarked on a journey of rigorous research and meticulous testing in order to identify the errors in our procedure. We, finally, concluded that our dosage of treatment was insufficient for the preferable transduction and should be increased. This was not solely due to a possibly reduced viral load within the exosomes but also to increased cellular stress on the day they underwent treatment. Furthermore, after receiving feedback from PIs and Lab experts regarding our results, we were informed that eGFP expression is not a reliable marker for such experiments, as it can use its indicatory ability throughout certain procedures.