iGEM 2024 — Team patras-med

{% endblock %}

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.

![Integrated Human Practices](https://static.igem.wiki/teams/5471/wet/engineer/circle-ezgif-com-crop.gif)

## 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.

1. [Cycle 1: Transfecting HEK293T](#cycle1)
2. [Cycle 2: Exosome Isolation](#cycle2)
3. [Cycle 3: Quantification of vexosome load](#cycle3)
4. [Cycle 4: Developing Testing Model](#cycle4)
5. [Cycle 5: Treatment/Transduction](#cycle5)

{% endblock %}

### miRNA strategy

The purpose behind the overexpression of a microRNA instead of
a protein for our gene therapy model is to target multiple
pathways and phenomena in the core of Alzheimer’s
pathophysiology. With this principle in mind, we chose the
**hsa-mir-195-5p** because of its targeting of virtually
all of AD’s pathological roots. In contrast to other microRNAs
that target neuronal inflammation and apoptosis (miR-34a), Αβ
amyloid plaque formation, and Tau aggregation (miR-132)
separately, mir-195-5p pushes back against all three of these
fronts simultaneously, essentially fighting AD to its core.

![miRNA](https://static.igem.wiki/teams/5471/teliko/path-graphic-teliko.webp)

### Transfer Plasmid Design Strategy

**Our transfer gene plasmid (pAAV-EF1a-mir195-eGFP-SV40pA)
([BBa\_K5471020](https://parts.igem.org/Part%3ABBa_K5471020)
)**
was carefully designed to accommodate the purposes of our
experiment. This plasmid is composed of the original pUC57
vector provided by Genscript, and Cassette 2 cloned into
position 2710. The reasoning behind its design is for it to be
usable as a transfer gene plasmid, which, along with pRepCap
and pHelper, will perform the triple transfection of HEK293T
cells and **produce rAAV2 viral particles** with the
desired properties. These properties include the
**simultaneous expression of mir-195 and eGFP**,
accomplished by Cassette 2. Additionally, due to the
properties of pUC57, this plasmid can independently
auto-replicate inside any cell and be selected later with the
use of the antibiotic Ampicillin.

![plasmid](https://static.igem.wiki/teams/5471/experimentals/transferegeneplasmid.png)

**Cassette 2 (ITR-EF1a-mir195-eGFP-SV40pA-ITR)([BBa\_K5471013](https://parts.igem.org/Part%3ABBa_K5471013))**
is an autonomous device for the simultaneous expression of
microRNA 195 and the eGFP reporter gene protein (Cassette 1 ),
specifically placed between a pair of ITR (inverted terminal
repeats) sequences for the AAV2 serotype ([BBa\_K5471004](https://parts.igem.org/Part%3ABBa_K5471004)). The ITR pair flanking makes the cassette ideal for rAAV
serotype 2 production because they enable the cassette they
flank to
**constitute the entire viral DNA (including the pair of ITR
sequences) of the produced rAAV2 particles**.

![wet lab](https://static.igem.wiki/teams/5471/wet/sxhma-wet-new-velakia.png)

**Cassette 1 (EF1a-mir195-eGFP-SV40pA)([BBa\_K5471012](https://parts.igem.org/Part%3ABBa_K5471012))**
is an
**autonomous device for the simultaneous expression of
microRNA 195 and the eGFP reporter gene protein**.

This cassette is comprised of (from the 5' end):

1. EF1a mir195 infused promoter, which is
**a new composite part** ([BBa\_K5471011](https://parts.igem.org/Part%3ABBa_K5471011),
[BBa\_K5471009](https://parts.igem.org/Part%3ABBa_K5471009),
[BBa\_K5471005](https://parts.igem.org/Part%3ABBa_K5471005))

2. a Kozak sequence ([BBa\_K5471006](https://parts.igem.org/Part%3ABBa_K5471006))

3. eGFP ([BBa\_K5471007](https://parts.igem.org/Part%3ABBa_K5471007))

4. SV40 polyadenylation signaling sequence ([BBa\_K5471008](https://parts.igem.org/Part%3ABBa_K5471008)) for the termination of transcription.

**EF1a Core Promoter**: The human eukaryotic translation
elongation factor 1 alpha (EF1a) promoter boasts strong gene
expression in most cell lines (including HEK293T), while being
more resilient and stable due to its mammalian origin,
compared to a viral-origin promoter that might fail to express
a gene or be silenced over time.

**EF1a Intron**: The human eukaryotic translation
elongation factor 1 alpha (EF1a) first intron is added
downstream of the EF1a core promoter to boost the gene
expression power of the promoter. This intron also provides an
ideal location to insert our “Gene of Interest.”

**The EF1a mir195 infused promoter** is a new composite
part responsible for
**driving the intragenic expression of mir195 inside the EF1a
intron while also ensuring the strong transcription of a
(eGFP) protein gene**. This new composite part provides the ability to efficiently
express microRNA 195 and simultaneously express a reporter
protein that confirms the expression of the microRNA.

![comppart](https://static.igem.wiki/teams/5471/experimentals/newcomppart.png)

This design **simulates the intragenic expression** of most
microRNAs by imitating the genomic locus where the hsa-mir-195
gene (87bp) is located.

![chrome3](https://static.igem.wiki/teams/5471/experimentals/chrom3.png)

![chrome4](https://static.igem.wiki/teams/5471/experimentals/chrom4.png)

The genomic locus chr17:7017465:7017851 (387bp) can be treated
as the “gene of interest” that is inserted into an already
existing cassette or plasmid vector. In our case, the 150bp
extra sequences flanking the hsa-mir-195 gene on either side
are identical to the ones that naturally flank the gene in the
human genome (150 + 150 + 87 = 387).

![genome](https://static.igem.wiki/teams/5471/experimentals/genomelocus195.png)

The goal of this faithful imitation of the human genomic locus
is to
**drive the natural transcription and modification
processes**
so that the mature hsa-mir-195-5p is produced inside the
treated cells. This method promises a stronger expression of
the mature microRNA, resulting in greater amounts compared to
other methods that insert the mature miRNA directly or use
shRNA mimics.

Specifically, the EF1a promoter used is borrowed from the
plasmid (scAAV GFP). Since we aim to insert a 387bp sequence,
it was crucial to
**preserve the parts of the intronic sequence near its 5’ and
3’ ends**. With this in mind, we decided to insert our “gene of
interest” at position 334 of the original EF1a promoter. The
combined length of this new part is 1178 bp, which is longer
than the original EF1a promoter, but similar in size to many
commonly used EF1a promoters. At the same time, the sequences
of the EF1a intron near the 5’ and 3’ ends remained untouched,
allowing the natural excision of the intron via cellular
mechanisms.

**eGFP**.The gene downstream of the new part could be any
protein, but we deemed a reporter protein gene more useful for
our experiments. We chose eGFP due to its
**relatively small size and strong fluorescence**. This
way, if fluorescence is detected, it strongly indicates that
the microRNA is also being produced.

**The Kozak sequence** (vertebrate consensus, a DNA/RNA
motif for strong initiation of translation, 1987) was placed
directly upstream of the eGFP coding region to achieve
**stronger and more stable initiation of translation**.

**The SV40 polyA signal**, or simian virus polyadenylation
signal, is responsible for signaling the
**polyadenylation of the 3' end of an mRNA transcript**.
Its role is to shield the mRNA from enzymatic digestion and
degradation inside the cell and to signal the termination of
the translation process. It was chosen for its
**small size (63bp), stability, and reliability**.

### Cell Culture

For the execution of our experiments, we selected
**adherent HEK293T cells**, a widely used derivative of the
HEK293 line (Human Embryonic Kidney cells) that has been
**transfected with the SV40 large T antigen**. This
modification enhances their suitability for transfection
protocols and gene therapy research. The combination of this
genetic alteration with the cell's high reproducibility and
ease of handling makes them an ideal choice for our initial
experimental workflow. This cell line was optimal for our
experiment for the reasons described above, in addition to the
fact that this cell line was more accessible and familiar to
our lab and their use would not compromize the
**safety** of our experiments, compared to the use iPSCs.

To ensure optimal preparation and cell viability, we adhered
to specific protocols for
**freezing, thawing, splitting, and seeding,** all of which
are detailed extensively in our experiment archive, as
documented in the lab notebook.

### Transfection

The construction of rAAVs began with the transient
**transfection of HEK293T cells** in culture using the
three essential plasmids: Transfer plasmid, Adenoviral Helper
plasmid, and Packaging plasmid. Three days later, we evaluated
our results by detecting **eGFP**, integrated within the
transgene plasmid, under a fluorescence microscope.

![transfection](https://static.igem.wiki/teams/5471/experimentals/transfection-2.png)

### Exosome Isolation

68 hours post-transfection, instead of proceeding with the
harvest of our vectors, we opted to
**isolate exosomes** from the culture supernatant, aiming
to capture the virus contained within them. To achieve this,
we **used exosome isolation reagent,** successfully
obtaining vexosomes, which were subsequently diluted in PBS
buffer. This isolation method was preferred to other methods
(ultracentrifugation), due to its
**high specificity and purity.**

![exosomoekkrisi](https://static.igem.wiki/teams/5471/experimentals/ekkrisi-exosomata.png)

### Measurement of Vexosome Load

After designing the exosome isolation procedure, we also aimed
to measure the
**quantity of rAAV encapsulated in the exosomes** to assess
the viral load, offering vital information for determining the
ideal dosage required for the transduction process.
Furthermore, this information is crucial for evaluating the
overall effectiveness of vexosome production, ensuring that we
achieve the desired efficiency and effectiveness in our
experimental results. To meet our objective, we selected
**qPCR** as the method for precisely quantifying the vector
load.

### Testing Model

Preparing our testing model presented several challenges.
After rejecting the final testing of our product in
Alzheimer's 3D cell culture due to the complexity of handling
and the elevated costs associated with the procedure, we
**turned to tau-expressing HEK293 cells**. Normally, HEK293
cells do not express tau proteins, which primarily stabilize
microtubules in neuronal cells. However, a specific
modification of their expression system allows HEK293 cells to
mimic the tau expression capacity of neuronal cells.

Unfortunately, due to technical and delivery issues, we were
unable to acquire these modified cells. As a result, we opted
to **utilize the HEK293T cells** already available and
assess the success of our therapy in relation to the
functionality of phosphatase PP2A, which is strongly and
directly linked to the dephosphorylation of Tau proteins.
Specifically, we decided to measure the effect of microRNA-195
on the methylation of the amino acid **Leucine 309** in the
catalytic subunit. As already mentioned, this methylation is
reduced in AD, and our goal is to
**upregulate the methylation** and, thus, the catalytic
activity of PP2A.

![pp2a](https://static.igem.wiki/teams/5471/experimentals/pp2a.png)

To better simulate the pathophysiology of Alzheimer's disease,
we also **incubated our cell culture with Calyculin A**, a
chemical compound known for its inhibitory effect on PP1 and
PP2A phosphatases. The diminished activity of PP2A in
Alzheimer's cells is associated with the increased
phosphorylation of tau proteins via
**qPCR and Flow Cytometry**
phosphorylation of tau proteins.

### Exosome modification

![rvg](https://static.igem.wiki/teams/5471/experimentals/rvg.png)

The next step in our experiment involves the
**incorporation of the RVG29 peptide onto the exosome
membrane**
through a click chemistry reaction, in order to achieve
specific targeting of neural cells. However, the absence of
neural cell culture, combined with the pressing timeline in
which the experiments were conducted, led us to decide against
testing this modification experimentally. Instead, we opted to
simulate it using Agent-Based Modeling (ABM), in Netlogo.

Click
[here](https://2024.igem.wiki/patras-med/model)
to read more about this.

![click](https://static.igem.wiki/teams/5471/experimentals/click-chemistry.png)

### Treatment/Transduction

Our exosomes were **administered to HEK293T cells**, that had
been treated with Calyculin A for 30 minutes, and incubated for
a duration of 72 hours, with the aim to facilitate the
transduction of these cells with the recombinant
adeno-associated virus (rAAV) that we had produced. The
transduction was assesed with the methods of
**qPCR and Flow Cytometry.**

![treatment](https://static.igem.wiki/teams/5471/experimentals/treatment.png)

### #WetLabJourney

![click](https://static.igem.wiki/teams/5471/wet/experiment-design/wet-lab-overview.png)

1. [miRNA strategy](#miRNA)
2. [Transfer plasmid design strategy](#Transfer)
3. [Cell culture](#Cell)
4. [Transfection](#Transfection)
5. [Exosome Isolation](#Isolation)
6. [Measurement of Vexosome Load](#Measurement)
7. [Testing Model](#Testing)
8. [Exosome modification](#Exosome)
9. [Treatment/Transduction](#Treatment)
10. [Wet Lab overview](#overview)

{% endblock %}

## 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

![results1](https://static.igem.wiki/teams/5471/wet/results/brightpicturecontrol.png)

![results2](https://static.igem.wiki/teams/5471/wet/results/darkpicturecontrol.png)

### TRANSFECTED

![results1](https://static.igem.wiki/teams/5471/wet/results/brightpicturetransfected.png)

![results2](https://static.igem.wiki/teams/5471/wet/results/darkpicturetransfected.png)

### 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.

![table](https://static.igem.wiki/teams/5471/wet/results/pcr-table.png)

**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.

![pcr](https://static.igem.wiki/teams/5471/wet/results/results-qpcr.png)

**FACS Results**

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

![t-ab]( https://static.igem.wiki/teams/5471/wet/results/t-ab.png)

![tc]( https://static.igem.wiki/teams/5471/wet/results/tcontrol.png)

![tt](https://static.igem.wiki/teams/5471/wet/results/ttrans.png)

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**.

1. [Results](#results)

* [Transfection Experiment](#transfection)
* [Titration of rAAV](#titration)
* [Treatment/Transduction experiment](#treatment)

2. [Proof of Concept](#proof)

* [Use of iPSCs](#iPSCs)
* [Plasmid Efficiency](#plasmid)
* [rAAV Production](#rAAV)
* [Modification of Exosomes](#modification)
* [Vexosomes as an Innovative Delivery System](#vexosomes)

{% endblock %}