Our Journey: firing the neurons leading to MS

Multiple sclerosis (MS) is a predominant disease “disease-personality” in neuroscientific research and the clinical world. For over 50 years, it has been a constant challenge for researchers and clinicians alike to unravel its well-hidden mysteries and uncover crucial treatment aspects of its pathophysiology. An underlying “flirt'' between the immune and nervous system intensifies itself to the extent that it takes on pathological dimensions, leading to a self-destructing love. “As a medical student”, Alexandros explained, “I've had numerous opportunities to learn about MS, mostly regarding current knowledge and progress already made. Then the notion struck, one that was absolutely and thoroughly fascinating: why not upend the current state of affairs by tackling novel therapeutically important aspects of MS etiology and setting ourselves apart from previously tested and established treatments? And all based on synthetic biology!” At this moment, the core motivation was established. NeuroMuSceteer was beginning to take shape!

Being a chronic illness of the central nervous system, the dynamic and unpredictable nature of MS presents a compelling challenge for medical research and patient care. MS leads to a range of physical and cognitive impairments. Despite its prevalence, public stigma and misinformation are not uncommon, often due to the lack of awareness and understanding. Therefore, bridging this knowledge gap and providing valuable insights through a new therapeutic strategy could be not only beneficial to patients themselves, but also their caregivers, and the community as a whole. Overcoming such a challenge could foster hope and encouragement among those affected by the disease. By shedding light on their daily struggles and triumphs, it could be possible both to inspire empathy and action as well as to “humanize” the complex parameters of the disease and the life it entails.

Yet, following our brainstorming timeline, “NeuroMuSceteer” was the last project proposal submitted. Prior to that, several remarkable ideas had already been discussed: an innovative point-of-care-testing (POCT) system for the early detection of Candida Auris; 'Conan,' a radiation-resistant bacterium as a key material for a novel space uniform design; two environmentally-focused projects addressing water pollution—one proposing a synthetic biofilter for efficient heavy metal and nitrogen-based compound collection, and the other suggesting a magnetic-bacteria biofilter to capture microplastics in the ocean. We also considered the idea of constructing 3D-printed organoids using cell cultures. Ultimately, there was an overwhelming consensus on the project's theme, though each choice was influenced by different criteria and reasoning.

9 REASONS WHY we chose this project

Local Solutions for Local Problems: Thessaloniki in the spotlight

Multiple sclerosis is a particularly pressing issue in our region, Thessaloniki, Greece, due to higher prevalence rates than in other parts of the country. According to studies, northern Greece, including Thessaloniki, has one of the highest MS incidences in Europe, most likely due to genetic, environmental, and lifestyle factors unique to the region ¹. This increased prevalence could be attributed to specific genetic markers found more frequently in northern Greek populations, as well as environmental triggers such as vitamin D deficiency ².

Furthermore, the Panhellenic Reference and Excellence Center for MS Research and Treatment lies in Thessaloniki, fostering pioneering research. The Second Neurology Department of AHEPA University Hospital ³ has been the cornerstone of this center. Nevertheless, addressing MS in Thessaloniki requires not only medical interventions but also public health strategies aimed at improving awareness, reducing stigma, and ensuring equitable access to care and support services. For the experiments and the implementation of our project, we were hosted at the Gene and Cell Therapy Center of George Papanikolaou Hospital in Thessaloniki, a GMP-certified facility for developing advanced gene and cell therapies in Greece. It constitutes the first facility of its kind in our country and its focus on CAR-T cells lately was a great inspiration for our project ⁴.

The Problem: where neurology meets immunology

Multiple sclerosis is unquestionably the most common non-traumatic disease affecting young people. The global incidence of MS is increasing (more than 2 million people), as is the disease's socioeconomic burden ⁵. Although the etiology is unknown, it is widely assumed that intricate gene-environment interactions play a substantial role and that the disease is autoimmune in origin. The disease is thought to begin with the activation of CD4+ T cells that target central nervous system (CNS) autoantigens and their infiltration into the CNS, followed by cytokine and antibody storms against oligodendrocyte and neuron antigens, which eventually lead to their irreversible destruction. MS pathology includes plaque formation in the CNS after inflammation and demyelination ⁶. Depending on which area(s) of the CNS is damaged, this can lead to a wide range of clinical symptoms.

Meeting CAAR: a cell therapy approach tailored to autoimmunity

CAAR T cells could be a new therapeutic strategy for autoimmune diseases, targeting and eliminating autoreactive B cells responsible for producing pathogenic antibodies. CAAR T cells have an extracellular domain derived from the autoantigen involved in the autoimmune response, which is linked to intracellular signaling domains that activate T cells when they bind to autoreactive B-cell receptors ⁷. Recent research has shown the effectiveness of CAAR T cells targeting desmoglein in mouse models of pemphigus vulgaris, aquaporin-4 in neuromyelitis optica, autoimmune encephalitis, NMDAR encephalitis and potential in treating systemic lupus erythematosus ^{8 9}. However, challenges such as off-target effects, potential cytokine release syndrome, and long-term safety concerns need thorough investigation. Additionally, the manufacturing complexity and costs associated with personalized cell therapies present significant hurdles for widespread clinical application ¹⁰.

CRISPR in the roster: making T regulatory cells

We sought to employ the CRISPR-Cas9 system to epigenetically induce expression of FOXP3, a transcription factor thought to be the defining characteristic of regulatory cells and necessary for their activation and survival, in order to lessen the potentially harmful side effects of our CAAR-T cells and to further suppress the autoimmune response. This would essentially turn our effector T cells into immunosuppressive Tregs, allowing us to retain immunological tolerance and improve therapy safety ¹¹.

T regulatory cells (Tregs) are essential for maintaining immune tolerance by suppressing autoreactive T cells through mechanisms like cytokine secretion (e.g., IL-10, TGF-β) and direct cell contact. In autoimmunity, impaired Treg function or numbers lead to the loss of immune tolerance, allowing self-reactive cells to attack healthy tissues.

CRISPR, an acronym for “Clustered Regularly Interspaced Short Palindromic Repeats”, functions as a precise gene-editing tool, leveraging a naturally occurring system in bacteria that serves as a defense mechanism against viruses. The CRISPR-Cas9 system, the most widely used variant, involves two key components: a guide RNA (gRNA) and the Cas9 enzyme. The gRNA is designed to match a specific DNA sequence within the genome, guiding the Cas9 enzyme to this exact location. Once bound to the target DNA, the Cas9 enzyme acts as molecular scissors, creating a double-strand break (DSB) in the DNA. The cell's natural repair mechanisms then kick in, and this is where genetic editing occurs ¹². Researchers can manipulate the repair process to disable a gene, insert a new gene, or correct a mutation. Undoubdedtly, this powerful technology is revolutionizing various scientific fields ¹³, however, the CRISPR-Cas-9 mediated introduction of DSBs has been associated with chromosomal translocations, chromothripsis and activation of apoptotic pathways[KP1]. Recently, epigenome editing has evolved as a safer and equally efficient approach. The epigenome editing tools consist of a DNA binding domain, such as deactivated Cas9 nuclease (dCas9) coupled with an epigenetic modifier domain (epigenome editor, epi-editor). Once the guide RNA (gRNA) successfully identifies and binds to the target genomic locus, such as a gene promoter or enhancer, the epi-editor is recruited to that specific site[KP2] . A broad range of epigenetic modifiers has been employed to either activate or suppress gene expression. One of the most well-known epigenetic activators is the dCas9-VPR[KP3], a synthetic transcriptional activation complex designed to strongly activate gene expression. It consists of three powerful transcriptional activators VP64, p65, Rta. Together, these activators form VPR, which significantly boosts transcriptional activation, much stronger than single transcriptional activators, when recruited to target gene. Unlike traditional CRISPR-Cas9, which introduces double-strand breaks to edit genes, dCas9-VPR modulates gene expression without altering the DNA sequence, thus reducing the risk of harmful mutations ¹⁴.

miRNAs in MS: some steps after the first date

Incorporating a remyelinating mechanism to the project design is crucial to combat the disease as a whole. After regulation of the autoimmune response against myelin, the damage that has already occurred could be repaired, and the disease progression would not only be decelerated but reversed. Myelin sheath restoration in the CNS, is the result of a cascade of steps that include the activation of stem cells called “Oligodendrocyte Progenitor Cells” (OPCs), their migration to demyelinated areas, their proliferation and finally, their differentiation to Oligodendrocytes (OLs), the mature cells responsible for myelin formation ¹⁵.

In multiple sclerosis lesions, while remyelination occurs initially -which explains remission periods-, the gradual accumulation of myelin debris and immune system cells inhibits it, mainly by blocking the differentiation of OPCs to OLs ¹⁶.

It should be highlighted that remyelination is one of the most sought-after targets for Multiple Sclerosis therapy, with the majority of research focusing on impaired OPC differentiation as the main culprit of failure. However, no such treatment is yet available ¹⁷.

MicroRNAs (miRNAs) are small, non-coding RNA molecules, typically about 22 nucleotides long, that play crucial roles in regulating gene expression. They bind to complementary sequences on target messenger RNAs (mRNAs), usually resulting in translational repression or target degradation and gene silencing ¹⁸.

The remyelination process in particular, involves a number of miRNAs in every stage that orchestrate OPC activity and can largely influence its success ¹⁹. That’s why miRNA delivery has been investigated as a remyelinating candidate for MS management yielding promising results ^{20 21}. Moreover, as expected, miRNA levels are altered in patients compared to their healthy counterparts, contributing to the physiological changes and allowing them to act as disease biomarkers ^{22 23}.

Combining those two functions, it is possible to utilize miRNAs in a singular theranostic mechanism that recognises commonly upregulated miRNAs in patient precursor cells and is then activated, leading to the release of miRNAs that boost cell maturation and remyelination.