Keeping an iGEM notebook was crucial for tracking the progress of our project, documenting each step and ensuring we stayed organized. It allowed us to capture key insights from expert consultations, helping us incorporate valuable feedback and refine our approach. By maintaining detailed records of all team discussions, we could easily reference past work and ensure consistency across tasks. Ultimately, the notebook became a vital tool for effective collaboration and troubleshooting, ensuring the project moved forward smoothly and systematically!
We initiated our first meeting with Ms. Boziki by presenting an overview of our project, NeuroMuSceteer, detailing its objectives
and potential impact. Following the presentation, she expressed interest in seeing more detailed information about the miRNAs and
provided comments on the following aspects.
c-ANCA: Peripheral cytokines play a significant role in systemic autoimmune diseases. However, in the case of multiple
sclerosis (MS), where the focus is on modifying oligodendrocytes, it is crucial to directly target the central nervous system
because this is where the primary effects occur.
IL-17, IL-23: Although these interleukins are targeted by monoclonal antibodies for treating systemic autoimmune diseases,
they do not have therapeutic indications for MS. In fact, inhibiting other cytokines which are beneficial in systemic autoimmune
diseases could potentially lead to excessive suppression in MS. Therefore, while modifying the immune system is important,
suppressing autoimmunity in the periphery does not necessarily translate to benefiting an autoimmune disease that affects the CNS.
Another topic for consideration was how mi-RNAs could act on oligodendrocyte cell lines and whether we would be using
commercially available options. Based on Mr. Aivaliotis’ description of the role of miRNAs, the specific actions that need to be
taken and the tests involved, we decided to use mixed cultures, as a pure oligodendrocyte one was not essential.
The question remains whether the selected molecules, such as interleukins and miRNAs, are the best possible targets. When
addressing interleukins within the CNS, it's crucial to consider the microenvironment. For instance, if we exclusively target
IL-17 –a cytokine produced by T-cells– we must acknowledge that, while activated T-effector cells are present in the CNS in cases
of MS, they are not the only contributors; Th1 cells and IL-23 are also prominent. This indicates that we need to carefully assess
what these modifications would specifically mean for oligodendrocytes in the CNS. Although cytokines are well-characterized in the
periphery, their effects on oligodendrocytes are less understood, making it essential to evaluate whether these targets are truly
the best choices.
Another question was raised about whether the treatment would be more effectively delivered to the CNS if
injected intrathecally into the subarachnoid space or directly administered into the bloodstream. In response, the experts
indicated that the effectiveness would depend on whether the product can cross the blood-brain barrier. They noted that
with an intrathecal injection there is a higher certainty that the product will cross and directly reach the target. On
the other hand, if the product is delivered through the bloodstream, a significantly larger quantity would be required.
Our first meeting with Ms Yiannaki and Ms Papadopoulou began with a comprehensive presentation of our project, NeuroMuSceteer. After reviewing our scientific
approach, the advisors suggested that the collection of T and B-cells should be performed from the blood rather than the cerebrospinal fluid (CSF), as it would
be easier and more practical to obtain a larger number of cells. Furthermore, they indicated that the re-administration of these cells through the CSF would
hold greater value, allowing them to act locally and more effectively. Additionally, they noted that it would be safer to utilize approaches that are currently
well-established and effective in both research and clinical settings. Ms Yannaki specifically recommended avoiding the use of nanoparticles due to the
significant challenges they pose. Instead, she proposed using CAR T cells, which can incorporate receptors with both extracellular and intracellular components
in order to redirect T-lymphocyte specificity. She also illustrated the potential of this technology with an example of targeting B lymphocytes and IL-17 with
a dual CAR receptor. Furthermore, she highlighted other cutting-edge technologies, including gene editing platforms (eg. CRISPR-Cas9) that have been successfully
applied in T-cell immunotherapy.
Following the discussions above, several questions arose that needed further consideration and investigation. One of them was whether to use the
microRNA-125a-3p as the experts indicated that increasing its levels might not be advisable, given its already high concentration in MS patients. Another
question concerned whether the plasmid we wanted to introduce would have transient expression. Considering that the initial plan was to inject the cells
locally, there was uncertainty about whether the cells could act locally for a sufficient time-period or not, before their activity ceases.
We started our meeting by discussing whether it is possible to modify the CAR T cells so that they are expressed in a way that attracts T cells to the area of
oligodendrocytes. For 4th generation CAR T cells, we need two signals to induce them; these could be a receptor on the damaged oligodendrocyte and IL-17.
CAR T cells have been found capable of crossing the blood-brain barrier. Therefore, we had to evaluate whether to administer these cells through injection
or explore an alternative approach.
During our conversation, T regulatory cells (Tregs) were brought up as they induce autoimmunity by destroying cytotoxic T cells and are a promising
option for treating autoimmune diseases. However, the experts stated that there isn't sufficient literature on their use for such purposes.
The final plan included the creation of 4th generation CAR-19 Tregs that simultaneously activate FOXP3. More specifically, we agreed on developing an
in vitro model, performing 4th generation CAR cell cloning and simultaneously conducting the epigenome editing of FOXP3 in a cell line. As 4th generation CAR
T-cells occur, they will also activate cytokines and the systemic toxicity in B and T cell lines will be prevented with their intrathecal administration.
Afterwards, the CAR receptors will be integrated into Tregs and we will perform initial cytometry to ensure the insertion of the receptor. Once this is
verified, we will proceed to assess their cytotoxicity. They also suggested we should explore ways to silence FOXP3 in cells to induce it epigenetically.
This approach minimizes the need for basic research, as B cell destruction is a well-established process, while the induction of FOXP3 via epigenetics offers
a novel and innovative strategy.
We initiated our meeting with Ms Boziki by discussing the theoretical foundation of our project and some recent modifications.
First of all, it was stated that oligodendrocytes are involved in only a portion of the MS pathogenesis. Given that they are responsible for the
myelination of axons, any targeted oligodendrocyte must be in a reasonable distance from the axon. Moreover, even if proximity is achieved, the oligodendrocyte
must have the ability to wrap around the axon and achieve remyelination. In terms of deliverables, we had outlined the following tasks: first, a literature
review to identify potential targets for constructing the transmembrane receptor, with a focus on interleukins and potentially a myelin oligodendrocyte
glycoprotein (MOG). The second one was an investigation into the biochemical pathways of oligodendrocytes, including their transcriptome and the alterations
in both within the context of multiple sclerosis.
Afterwards, an emerging question occurred regarding the targets that would be beneficial in oligodendrocytes. The experts stated that both primary
and model cells could be used in laboratory terms, giving us the flexibility to choose our cellular line. However, achieving measurable responses might be
challenging, if not impossible, due to the complex communication pathways between target cells and other cells in the brain's environment. Given that our
initial focus was solely on laboratory application, we could start with a model expression that we know will work in the lab, even if we have no indication
that it will be effective in patients. Moreover, Ms Boziki’s research team mentioned the possibility of working with patients' peripheral blood samples,
provided we obtain informed consent.
In conclusion, the experts fully supported the miRNA aspect of our project and, although they expressed reservations regarding the functionality of
the membrane receptor approach, they tentatively agreed to let us work on designing it.
Ms. Yannaki suggested using viral vectors, but she had reservations about their practical aspect, noting that they haven't been particularly favored for
delivering CARs. In contrast, Ms Papadopoulou proposed either employing a dual CAAR receptor or using two separate viral vectors to transduce the same cells,
resulting in CAARs that target both MOG and MBP within the same product. Alternatively, we could create two different products: one targeting MOG and the other
targeting MBP. Regarding the technical challenges and effectiveness of the initial idea, its viability can only be determined through comparative experiments
and cannot be predicted in advance. Therefore, we decided to proceed with MOG and consider MBP later on.
Instead of working with T cells, we considered using HEK 293 cells and a cell line that simulates T cells.
According to Ms. Papadopoulou, we might not have time to integrate the CAAR into the cells, but we could do the cloning and for the FOXP3 component,
we could express and introduce it into a cell line. We also had to determine whether the CAAR sequence would be taken as a whole or in two parts, a decision
that required careful consideration and a good understanding of the Golden Gate Assembly. The vector we decided on was Lenti-PGK, from which could remove the
GFP or the PGK insulator in order to increase the safety of the lentiviral vector (which might make Sleeping Beauty unnecessary).
Ms. Psatha proceeded on sending us the map of the insulated PGK-GFP plasmid with a C1 area modification and our next step was to figure out the CAAR
receptor’s insertion.
We started our meeting by highlighting that the main goal of our project is the reduction of inflammatory relapses in MS patients, which will be achieved by
differentiating Tregs with an integrated CAAR receptor that carries an extracellular autoantibody against MOG.
Afterwards, we discussed the mechanism that would promote the demyelination of oligodendrocytes, noting that we will probably administer the particles
intranasally so they reach the CNS without crossing the blood-brain barrier. At that point of our research, we had decided on four miRNAs, two of which have
been proven to promote remyelination and two that promote hybrid chain reaction (HCR). In this context, Ms Boziki asked whether a sufficiently broad range had
been studied in order to ensure that selected miRNAs are the most representative and stated that an adequate sample size might be 20-30 miRNAs. However, she
suggested that we first apply the delivery system and then test the remyelination model in order to confirm that the number of oligodendrocytes indeed increases.
She also requested evidence supporting the selection of miRNAs, including their origins (clinical samples of MS patients, experimental models or cell cultures),
and encouraged us to seek answers from in vitro studies, as they would also be acceptable.
To summarize, she suggested evaluating the use of a myelin basic protein (MBP), as MOG35-55 works well in animal models but encounters issues with
major histocompatibility complexes and machinery in humans or human models. Given these challenges, we had to either examine another antigenic epitope or
continue with MOG, knowing that our project might never advance to human trials.