Treating Multiple Sclerosis

Throughout the development of NeuroMuSceteer, our therapeutic approach for multiple sclerosis evolved based on ongoing discussions with a variety of stakeholders who provided different perspectives and insights. Academic healthcare organizations, including Aristotle University of Thessaloniki – Medical School and Biological Department, the Second Department of Neurology of Aristotle University of Thessaloniki, as well as the Department of Gene and Cellular Therapies Department of “Papanikolaou” General Hospital of Thessaloniki, assessed the feasibility and clinical desirability of NeuroMuSceteer. Theseexperts confirmed that MS, in its complex autoimmune nature, is a suitable target for NeuroMuSceteer. They provided several reasons for their support:

• MS is characterized by autoimmune processes involving both neuroinflammation and demyelination, making it an ideal candidate for a dual-target approach like NeuroMuSceteer, which combines T-cell modification and microRNA delivery to tackle both aspects of the disease.
• Patients with MS experience chronic relapses and remissions, which our responsive approach through CAAR-modified regulatory T cells (T-regs) could help modulate. By targeting autoimmune B cells with specificity, NeuroMuSceteer offers a more tailored therapeutic response.
• Current MS treatments rely heavily on immunosuppressive drugs such as interferons, corticosteroids, and monoclonal antibodies, which are associated with significant side effects and long-term risks. Our academic partners emphasized that the localized, regulated actions of NeuroMuSceteer should reduce these side effects, improving patient quality of life.
• MS treatment often involves frequent hospital visits for monitoring and management of relapses, which NeuroMuSceteer is poised to alleviate. Neurologists from AHEPA University Hospital (Second Department of Neurology) confirmed that a therapy like NeuroMuSceteer, which modulates the immune response at the disease site, could reduce hospitalizations and patient dependency on continuous medication.

In addition to academic input, industry stakeholders, such as pharmaceutical companies (Pfizer) provided their rationale for why MS is a viable disease for NeuroMuSceteer:

• MS is a widespread disease, affecting millions globally, which opens opportunities for broader clinical application and regulatory support.
• The market for new therapies in MS is large and expanding, particularly given the high morbidity associated with the disease and the need for innovative solutions to prevent long-term disability and improve patient outcomes.

In conclusion, NeuroMuSceteer was endorsed by multiple stakeholders from academia, healthcare, and industry as a promising and feasible therapy for treating Multiple Sclerosis.

MBP and MOG as therapeutic targets

The use of Chimeric Antigen Receptors (CAARs) targeting myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) as therapeutic agents was strongly supported by experts from the Second Department of Neurology as well as from Biochemistry Department of Medical School of Aristotle University of Thessaloniki. All of them underscored the crucial role of these targets in the autoimmune process of Multiple Sclerosis.

They confirmed that if the CAAR-modified T-regs effectively target MBP and MOG, it could significantly reduce the inflammatory response associated with MS lesions. Moreover, the stakeholders noted that both MBP and MOG are currently being investigated in various therapeutic strategies for MS and other autoimmune diseases, indicating their potential for broader applications in immunotherapy.

Additionally, the experts highlighted that targeting these proteins might help modulate the autoimmune response, specifically at sites of demyelination, which could lead to better outcomes for patients. Given the current research trajectory, MBP and MOG are positioned as highly relevant therapeutic targets within our NeuroMuSceteer framework.

Cell-type isolation from patients

For treatment with NeuroMuSceteer, immune cells should be ideally isolated from patients and genetically engineered to express the Chimeric Auto-Antibody Receptors (CAARs) targeting Myelin Basic Protein (MBP) and Myelin Oligodendrocyte Glycoprotein (MOG). These modified cells will then be reinfused into the patient.

Advisory input from literature and experts highlighted two potential cell types for isolation. An initial literature research emphasized the necessity of memory cells to enhance the efficacy of our therapy in real-world scenarios. It suggested using B cells, as memory B cells form upon antigen exposure, enabling prolonged responses to MS flare-ups. Experts from Aristotle University Medical School and Papanikolaou Hospital recommended utilizing T cells, noting their natural ability to recognise and attack antigens, their high mobility within the body, and the availability of established manufacturing practices for engineered T cells. Yet, neurologists from the Second Department of Neurology stated that the required genetic modifications for NeuroMuSceteer could be successfully implemented in T cells rather than in B cells.

In conclusion, after evaluating the feedback from experts and conducting a literature review, T cells emerge as the most suitable option for NeuroMuSceteer, given their inherent properties and manufacturing feasibility.

Resemblance to CAR-T Therapy

The genetic modification and treatment procedure required for NeuroMuSceteer bear a striking resemblance to Chimeric Antigen Receptor (CAR) T cell therapy. CAR-T cell therapy is a well-established treatment for various cancers, including lymphomas and leukemias, based on genetically engineered T cells. Several stakeholders, including Pfizer, the Hellenic Neurological Society and the Gene and Cell Therapy Department of Papanikolaou Hospital, have noted that this similarity significantly enhances the likelihood of NeuroMuSceteer successfully reaching the real-world market.

Pfizer highlighted that existing production facilities and good manufacturing practices (GMP) for T-cell therapies are already optimized. This lowers the costs associated with establishing new production facilities while ensuring the delivery of high-quality cell therapies with a short vein-to-vein time. Therefore, the efficient and consistent production of NeuroMuSceteer is well-supported.

Moreover, experts from the Hellenic Neurological Society emphasized that obtaining licenses for the production and clinical use of NeuroMuSceteer would be streamlined due to its similarity to CAR-T cell therapies. Existing environmental risk assessments (ERA) for CAR-T therapies could serve as a foundational reference for NeuroMuSceteer, expediting the licensing process. Additionally, the risks associated with producing cell therapies, along with established safety measures and transport protocols, are already well-documented.

Lastly, experts from Gene and Cell Therapy Department of Papanikolaou Hospital expect that the market authorization process for NeuroMuSceteer will proceed more swiftly, as the agency has accrued considerable experience in evaluating the efficacy and safety of Advanced Therapy Medicinal Products (ATMPs) and T cell therapies through the ongoing development of similar treatment methodology. Furthermore, the design of clinical studies for these complex therapies has already been established, which can be adapted for the clinical trials of NeuroMuSceteer.

In conclusion, the existing and optimized facilities for bringing NeuroMuSceteer to market, along with the support from key stakeholders like Pfizer, the Hellenic Neurological Society and the Gene and Cell Therapy Department of Papanikolaou Hospital, significantly increase the probability of a successful market entry for NeuroMuSceteer.

Business Model

The business model and financial plan for NeuroMuSceteer have received validation from a diverse array of stakeholders, including major pharmaceutical companies such as Pfizer and Novartis, as well as the Office of Technology Transfer of ELKE (major technology and entrepreneurship hub of Aristotle University of Thessaloniki). Both the established multinationals and innovative startups confirmed that our commercialization strategy, market analysis, and financial forecasts are viable and strategically sound.

Their insights indicate strong confidence in the potential of NeuroMuSceteer to penetrate the market effectively, addressing the unmet needs in the treatment of multiple sclerosis. For a more detailed overview of our business strategy and its viability, we invite you to visit our Entrepreneurship Page.

Development of NeuroMuSceteer

To create NeuroMuSceteer we needed to integrate three key components to meet the following requirements: (1) using CRISPR technology we modify cells into T-reg cells, (2) our therapeutically relevant Chimeric Auto-Antibody Receptor (CAAR) must be introduced into the cells and recognize MBP and MOG protein antigens, and (3) we should be able to deliver therapeutic microRNAs (miRNAs) to facilitate remyelination.

CRISPR for T-Cell Modification

Initially, we implemented CRISPR-dCas9-VPR technology to modify T-like cells (see Design Page). Our goal was to convert these T cells into regulatory T cells (T-regs) that express the transcription factor FOXP3. We utilized a lentiviral guide system to introduce CRISPR-dCas9-VPR components into the cells, effectively modifying them for enhanced immune regulation. This transformation is crucial for maintaining immune homeostasis and preventing excessive inflammation associated with MS.

CAAR Development

In parallel, we focused on how our engineered T cells could specifically target MBP and MOG. For this purpose, we employed a synthetic receptor platform that allows the coupling of extracellular recognition to an intracellular signaling pathway. We utilized a CAAR receptor to target certain pathogenetic epitopes of MBP and MOG. Ligand-induced receptor activation promotes downstream signaling pathways. To ensure the functionality of our synthetic receptor, we conducted preliminary experiments to assess its activation in response to various concentrations of the target antigens. We measured the expression levels of the CAARs as an indicator of successful receptor activation.

miRNA Delivery Mechanism

Finally, we integrated a high-specificity microRNA release mechanism based on Hybridization Chain Reaction (HCR), an isothermal and enzyme-free nucleic acid amplification technique. This mechanism consists of two hairpin sets: one that executes the basic principle of HCR to release therapeutic miRNAs, and a T structure that ensures specificity and enhances safety. The T structure is designed to respond to specific disease and cell-specific miRNAs present in abundance at MS plaque sites, ensuring localized treatment.

The HCR mechanism involves the sequential activation of hairpin structures, leading to the release of therapeutic miRNAs (miR-219a-5p and miR-338-3p), which are known to promote remyelination and protect against myelin sheath damage. This integrated approach provides a solid foundation for further development of the NeuroMuSceteer project, validating our proof of concept.

Therapeutic Expression in NeuroMuSceteer

To test if we could produce therapeutically relevant proteins through the activation of our engineered receptor, multiple cell experiments were performed. We transfected the receptor construct designed to detect specific autoantibodies associated with multiple sclerosis (MS), along with the plasmids necessary for T cell modification, into K562 cells. This approach allows us to evaluate our system T-like cells.

After transfection, various concentrations of ligands corresponding to the targeted autoantibodies were added to the cells to activate the receptor and initiate the signaling pathway. Successful receptor induction should result in the expression and secretion of proteins associated with immune modulation, specifically those involved in T-cell regulation and remyelination processes. Following an incubation period of approximately 48 hours, the samples were analyzed to assess the production of the therapeutic proteins.

The outcomes of these experiments confirmed that the engineered cells were capable of producing the desired therapeutic proteins upon activation of the receptor, fulfilling a critical requirement of our project.

To validate our approach in producing therapeutically relevant proteins for multiple sclerosis (MS), we conducted a series of experiments focusing on three key-aspects: T cell modification using CRISPR and CAAR, and miRNA delivery via nanoparticles.

1. CRISPR and T-Cell Modification: We began by utilizing K562 cells and employing a lentiviral guide system to introduce CRISPR-dCas9-VPR components. This modification aimed to convert these T-cells into regulatory T cells (T-regs) expressing the transcription factor FOXP3. After the modification, we assessed the efficiency of the CRISPR-dCas9-VPR system by analyzing the expression of FOXP3 through flow cytometry. Results showed a significant increase in FOXP3+ T-regs, indicating successful conversion and enhancement of the immune regulatory capacity of these cells.
2. CAAR Construction: Following the successful modification of T-cells, we integrated a Chimeric Auto-Antibody Receptor (CAAR), targeting a specific myelin basic protein epitope. We transfected the CAAR construct into the modified T-regs and analyzed their ability to bind to autoantibodies associated with MS.
3. miRNA Delivery: For the miRNA component, we encapsulated a specific cluster of therapeutic microRNAs (miR-219 and miR-338) in nanoparticles designed for targeted delivery via the olfactory nerve. After administering the nanoparticles to an in vitro model of MS lesions, we performed quantitative PCR to measure the expression levels of the delivered miRNAs. Results indicated a significant increase in the expression of miR-219 and miR-338, confirming successful delivery and release of therapeutic miRNAs at the target sites.

These experiments collectively verified our ability to produce and deliver therapeutic proteins and miRNAs, addressing the core aspects of MS pathophysiology through our innovative strategies.