Product Development

1. A modular system to easily build CAARs (autoantibody receptors) and clone new antigen epitopes
2. an AI interface for selecting the best promoters and ribosome binding sites(text)
3. a protocol for miRNA target selection
4. and software for the design of the oligos that will assist in the selective, personalised tissue regeneration

Constructing a Business Plan

As an aspiring business, it is imperative to develop a solid plan that outlines our goals, the steps we have to take and our place in the current market. The figure above presents the key components of a business plan as listed by the EU Institute of Entrepreneurship Development ¹. Our immediate goal was to start gathering important information about our product and the industry. To do that, we put together a list of relevant stakeholders that would help us fill in the gaps in our knowledge and improve our analyses;

• AUTh Technology Transfer Office
• AUTh Biomedical Research and Education Special Unit
• Industrial Property Organization
• National Organization for Medicines and European Medicines Agency
• National Organization For Health Care Services

In our Entrepreneurship Page, we describe in great detail all the work we did to lay a strong foundation for our next steps, protect our intellectual property while actively driving NeuroMusceteer to the pharmaceutical market.

Cell Production

The production process involves a series of steps,throughout which stringent quality control measures are applied to ensure the cells meet safety and efficacy standards. Advances are being made to optimize this process, including using closed, automated systems to improve scalability and consistency.

Collection (Leukapheresis): The process begins by collecting the patient's T cells from peripheral blood through leukapheresis. This separates the mononuclear cells (including T cells) from the rest of the blood, with the uncollected components being returned to the patient.

Cell Activation: Once in the facility, the T cells are activated using anti-CD3 and anti-CD28 antibodies. These are either immobilized on beads or other substrates to stimulate T-cell receptors and costimulatory signals, which is necessary for the cells to proliferate and accept genetic modifications.

Gene Transfer (Transduction or Transfection): The activated T cells are genetically modified to express a chimeric autoantibody receptor (CAAR) as well as the FOXP3 gene that turns them into Tregs.

Expansion: The genetically modified T cells are cultured and expanded in bioreactors or gas-permeable culture systems. During this step, cytokines such as IL-2, IL-7, or IL-15 are often added to promote T-cell growth and increase the number of cells to reach the desired therapeutic dose.

Testing: The final CAR T cell product undergoes a series of tests to ensure it meets safety and potency standards. This includes tests for sterility, identity, viability, and functional potency (such as the ability to kill cancer cells in vitro).

Cryopreservation and Shipping: After passing quality control, the CAR T cells are typically cryopreserved and shipped to the clinical site in liquid nitrogen shippers.

Infusion: At the clinical site, the CAR T cells are thawed and infused back into the patient. This infusion is usually followed by monitoring for side effects, such as cytokine release syndrome (CRS), which can occur due to the activation of the immune system.

Storage

Cryopreservation

Cryopreservation of cells requires suspending them in a specialized solution containing cryoprotective agents (CPAs) to help them withstand the stresses associated with freezing and thawing. Commonly used CPAs include dimethyl sulfoxide (DMSO) and glycerol ^{3 4}.

Cooling rate:

The cooling rate, from the solution's freezing point to around −50 °C, significantly affects post-thaw cell recovery. Controlled-rate freezing ensures optimal survival by maintaining the ideal cooling rate.

Storage Conditions:

Frozen cell therapy products should be stored at temperatures below −150 °C for long-term stability. Minimizing transient warming during storage access prevents degradation and preserves cell quality. 

Storage should be done in vapor phase nitrogen tanks for up to approximately 6 months. Hospitals with a cell processing facility, or other cryopreservation premises, preferably have multiple nitrogen tanks for back-up, as well as separating CAR-T cells with market authorization to be distinguished from those used in trials. A main filling reservoir supplies the tanks, with the refilling process fully automated and real-time level tracking in place. A continuous alert system should be triggered when levels drop below a certain point. Additionally, temperature trends must be consistently tracked and regulated.

Thawing Conditions:

Thawed samples are often warmed in a 37 °C water bath to ensure proper cell viability. New thawing devices improve safety and consistency, with thawing rates influenced by the prior cooling rate.

Post-Thaw Processing:

After thawing, cells can be infused directly, diluted, or washed to reduce osmotic stress. Post-thaw processing steps are critical for determining the final product administered to the patient.

The above process is necessary when cell products are not produced on site or for immediate use. Although slightly less potent than their fresh alternative, cryopreserved CAR-T cells have proven to be a perfectly viable option that allows spatio-temporal flexibility ⁵.

Hairpin Storage

Like cell therapy, ideally our hairpins would be constructed on site and swiftly delivered to the patient. However, if needed, storage at -20oC is possible for either hairpins or individual oligos that would be hybridized in an on-site lab. The latter would last 6 months in water and up to a year when lyophilised ^{6 7}.

Delivery

(a) Locoregional delivery of CAAR-Tregs

Region-specific delivery in the central nervous system (CNS) is an emerging strategy aimed at improving the efficacy of immunotherapy for CNS conditions, particularly gliomas and glioblastoma multiforme (GBM). It is highly probable that traditional systemic delivery of CAAR-Tregs would face challenges such as poor T cell infiltration due to the blood-brain barrier. On the other hand, locoregional approaches, including intracavitary and intraventricular delivery would allow for direct administration of our engineered T cells to MS lesions. This method enhances therapeutic concentration while minimizing systemic toxicity, and early clinical trials suggest it is both feasible and safe​ ⁸. The administration would be done only by trained professionals via devices such as the Ommaya Reservoir, where the drug is stored in a dome-shaped reservoir that connects with a catheter and delivers it to a specific region ⁹.

(b1) Nanocarrier delivery of miR release mechanism

The delivery of the mechanism is a determining factor of its in vivo activity and thus, a critical component of the overall design. A carrier should be able to successfully cross the cell membrane, provide sufficient drug bioavailability and precise release without inducing antigenicity or other toxic effects. Nanocarriers fulfill the criteria and have been already used and studied as molecule transportation systems to the brain. There are plenty of nanocarrier subtypes with different properties as described below ^{10 11}.

Nanocarrier-based miR delivery for multiple sclerosis, specifically for miR-219, has been already investigated ¹². The researchers compared the efficacy of polymer and liposome vectors –already approved and in use– to that of extracellular vesicles. Even though they observed higher uptake by OPCs when using liposomes and polymer nanoparticles, induction of differentiation and clinical improvement only occurred with EVs which demonstrated their feasibility and promising future application. Given that oligodendrocytes are a known source of Extracellular Vesicles in the Central Nervous System, their utilization as molecule carriers to OL lineage cells seems like an option worth exploring ¹³.

(b2) Nose-to-brain nanocarrier administration

To reach the CNS and execute their function, drugs should be able to cross the blood-brain barrier, which often poses a major hurdle. However, in recent years an alternative method capable of bypassing the barrier has sparked interest and proven to be quite beneficial; intranasal delivery, also referred to as nose-to-brain. The direct transportation from the nasal cavity to the brain is possible via either the trigeminal or the olfactory nerve and taking the limited available surface into account, it is best suited for nano-sized formulations ¹⁴. Nose-to-brain administration is also non-invasive, safe and shows higher levels of patient adherence. On that basis, numerous established multiple sclerosis medications displayed favorable results when delivered intranasally to animal models and clinical trials ^{15 16 17 18 19}. In spite of existing validation data, the nose-to-brain delivery of nanomedicines for multiple sclerosis treatment is still in the early stages of its development with many unresolved parameters, notably insufficient accumulation to action-site and dose calculation. Nonetheless, it shows immense potential and greatly compliments our approach in its entirety.

Safety Concerns and Management

CAAR-Tregs

Although our CAAR-Tregs show great promise for autoimmune treatment, there are significant adverse effects they may cause. The most dangerous of these is cytokine release syndrome (CRS). CRS results from a massive release of cytokines following T-cell activation, often leading to fever, hypotension, and hypoxia. Severe cases can evolve into life-threatening conditions like hemophagocytic lymphohistiocytosis (HLH). Therefore, although transforming effector cells to regulatory decreases the chances for CRS we have to consider a management plan.

Management of CRS depends on severity. Mild cases are managed with supportive care, while severe cases may require cytokine antagonists and corticosteroids. Brain imaging and prophylactic antiseizure medication are also recommended.

Other adverse effects include cytopenias and B-cell aplasia, which increase the risk of infections. Regular monitoring and infection prophylaxis are crucial in managing these complications​ ²⁰.

miR treatment

miR-219

miR-219 is involved in a plethora of pathways which inevitably links it to some pathogenic effects. It was discovered to promote the progression of hepatocellular carcinoma by inhibiting the expression of a suppressive oncogene CDH1 ²¹. Furthermore, overexpression of miR-219 downregulated TMEM98 -an important proliferation and migration factor of keratinocytes- and therefore hindered wound healing ²². By targeting the LRH-1 gene of the Wnt/β-catenin signalling cascade and reducing cardiomyocyte apoptosis, it was also shown to take part in the pathology of Congenital Heart Disease ²³.

miR-338

Like miR-219, miR-338 is a regulator of various genetic elements, sometimes with negative impact. As an inhibitor of PIK3C3, it boosts tumour metastasis and acts as a marker of poor prognosis of colorectal cancer progression ²⁴. A clinical study indicated that increased expression of miR-338 correlated with insufficient treatment response in acute myeloid leukaemia patients ²⁵. Most importantly, heightened expression of this molecule was found to suppress Treg function in pemphigus vulgaris, an autoimmune disease, by blocking the expression of the RUNX1 gene and therefore the essential FOXP3 transcription factor ²⁶.

The above findings do not negate the selection of miR-338 and miR-219. Despite the severity of the effects mentioned, it should be noted that they concern miR upregulation. On the contrary, the treatment in question aims at the controlled restoration of the molecules’ levels which are diminished in Multiple Sclerosis patients. Additionally, the pathogenic interactions do not involve the Central Nervous System, but other tissues such as the heart and large intestine. On that end, optimizing our delivery and administration methods would prevent off-target release and in combination with constant patient monitoring, minimize any potential adverse response.

Authorization

To be authorized as a therapy, NeuroMuSceteer would need to go through several phases of clinical trials evaluating various pharmacodynamic and pharmacokinetic properties in both healthy participants and patients. Then, its manufacturers would need to submit an MAA (Marketing Authorization Application) to the regulatory authority that monitors the market they want to reach.

We studied the characteristics and duration of clinical trials worldwide as well as the MAA approval procedure of the EMA, the administrative body responsible for pharmaceuticals in the EU. Below are brief presentations of said proceedings, indicating a 5-year timeline for NeuroMusceteer, from entering clinical trials to reaching patients. Taking into account the necessary research and preclinical trials that are still required beforehand, we predict an 8-10 year period from now until authorization.