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Implementation

Overview

  • Introduction
  • Design
  • Mesenchymal Stem cells (MSCs) role in immune regulation
  • CRISPR systems integration with MSCs
  • Safety
  • Pre-clinical studies
  • In vitro Validation of the therapeutic approach:
  • In vivo validation by animal model
  • Clinical trial
  • Market authorization
  • Step one:
  • Step two:
  • Step three:
  • Step four:
  • Real World Implementation
  • Route of administration
  • End users
  • Future plan
  • Challenges
  • References
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    Image illustrating how photothermal therapy.
    Fig1. The project steps from the lab to the world.

    Introduction

    As our project is coming to an end to enlighten the medical field, we -AFCM Egypt- found it essential to think about how our 2023 project will be implemented in the real world, what its future applications are, and what its distribution and logistics are. Considering that our end users are patients, we have first to give an overview of the disease we are targeting. Furthermore, we have to talk about the prevalence and the incidence of this disease because they are some of the crucial factors that cause us to choose this specific autoimmune disease. In January 2023, our team was appointing a meeting to come up with an idea to work on this year. We found that one of the leading causes for work interruption in Egypt was due to workers' holidays and this leads to hindering the development of the country. One of the most common causes for workers' sick leave was due to joint pain. Eventually, we found that joint pain can be either inflammatory or degenerative, and rheumatoid arthritis, one of the inflammatory autoimmune diseases, has a high prevalence in Africa. Thus, we decided to work on a complete cure for rheumatoid arthritis.

    Rheumatoid arthritis(RA) is an autoimmune disease, which means that the body’s immune system attacks its healthy cells. Moreover, it is a multi-factorial disease which means it has no specific etiology to target through drugs. It has many predisposing factors such as genetic predisposition, smoking, stress, gender, obesity, and age. As an inflammatory disease, RA not only affects joints symmetrically but also has the same symptoms and signs of any inflammatory arthritis: morning stiffness of the affected joints, pain, swelling, weakness, fatigue, and weight loss. It also causes extra-articular manifestations such as anterior uveitis, pulmonary fibrosis, pleural effusion. The RA regimen among the guidelines is mainly supportive, not definitive. Indeed, clinicians prescribe mainly corticosteroids to decrease inflammation and immunity. Second lines of treatment may include immunosuppressants and Disease-modifying antirheumatic drugs (DMARDs) as methotrexate and biological therapies. On the other hand, our approach is the complete cure of RA through mesenchymal stem cell-based therapy. Mesenchymal stem cells (MSCs) are stromal cells that can self-renew and also exhibit multilineage differentiation. We are developing SUPER-MSCs: Smart, Universal, Pleomorphic, Endogenous, Regulator of immunity which eradicate the autoreacive B-cell in 2 stages. The process includes two main stages:
    1- Firstly, a recognition stage, where our engineered MSCs specifically bind to the specific auto-reactive B-cells that produce the Anti-Citrullinated Protein Antibody (ACPA), which is the main antibody implicated in the pathogenesis of RA. This is done by designing a synthetic receptor called the (Syn-Notch) which is engineered to be produced on the surface of our MSCs.
    2- Secondly,Upon recognition of the auto-reactive B-cells, our SUPER-cell produces engineered exosomes targeting auto-reactive B-cells and attacking them, consequently lowering their numbers, and reducing the severity of the patient’s condition.

    This protects the patient from the side effects of corticosteroids and Immuno-suppressants: infections, osteoporosis, hypertension, hyperglycemia. (4)

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    Fig 2. Illustration describes the progression of rheumatoid arthritis diagnosis and treatment.

    Design

    Mesenchymal Stem cells (MSCs) role in immune regulation

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    Fig 3. Shows the possible sources of the MSCs.
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    Fig 4. Illustration shows the innate function of MSCs in immune regulation by: Increasing T-regulatory proliferation, Inhibition of Natural Killer(NK) proliferation, and inhibition of T-cell proliferation. It also has regenerative functions through Angiogenesis, Neuro-protection, Self-replication, and differentiation.

    CRISPR systems integration with MSCs

    CRISPR system is one of the most important parts of our project as we used it to induce apoptosis of auto-reactive B-cells. We designed it to be targeted to the survival signal inside the B-cell activating factor receptor (BAFFR) that is vital for the survival of B cells. This CRISPR system will be specific only for the auto-reactive B-cells by our multi-layer safety system that will be discussed later in the safety part of the page.

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    Fig 5.This figure illustrates our CRISPR system designed to be specific for auto-reactive B-cells
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    Fig 6. Shows the 3 safety layers we designed for our approach: Syn-Notch receptor, Exosomal receptor, Safety switch

    Safety

    To make our SUPER MSCs safe and to prevent any potential adverse effects, we developed a multi-layer safety system to take its benefits and prevent its harms:

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    Fig 7. shows the structure of Syn-Notch receptor

    • The 1st layer is Synthetic notch receptor → Syn-notch receptor is composed of 3 domains Internal domain, Transmembrane domain, and External domain. The internal domain is responsible for the internal signal that will activate a cascade of reactions that leads to the production of our engineered exosomes that carry the CRISPR system. The transmembrane domain fixes the receptor in the cell membrane and it is almost the same in all Syn-notch receptors. The external domain is the most important part as it is responsible for the specificity of the receptor by designing it to have a high affinity for B-cell receptor (BCR) which is considered specific for each B-cell colony. This will make the activation of the system conditioned by the presence of auto-reactive B-cell colony.

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      Fig 8.Displays the Function of exosomal receptor.

    • The 2nd layer is Exosomal receptor → After activation of the Syn-notch receptor, the exosomes will be produced in the media. Moreover, ensuring that they will enter the auto-reactive B-cell, we designed them with a receptor specific for the auto-reactive B-cells receptor that will increase the specificity for the approach as a whole.

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      Fig 9. Displays the function of the safety switch based on ADAR enzyme.

    • The 3rd layer is Safety switch → By the delivery of the CRISPR system, there are some regulations to follow: It will be under regulation of a specific switch called DART VADAR switch that is designed to be sensitive to the mRNA of the ACPA. By using this switch, we will prevent activation of the CRISPR system inside other normal cells.

    Pre-clinical studies

    In vitro Validation of the therapeutic approach:

    In our approach, we needed to validate each sector separately as follows:

    • Structural validation to ensure that the biological parts were correctly expressed to validate the expression of the Syn-notch receptor that was tagged with a Flag tag in our design to be detected by anti-flag-tag antibodies to ensure its expression on the cell membrane.Furthermore, the engineered exosomes will be tagged by His tag and then will be detected using anti-His tag antibodies to ensure that they will be secreted.

    • In addition to the structural validation, we will also perform a functional validation that ensures that the expressed part is doing its proper function. During this step, we will test the apoptotic function of our system to ensure that our system has the ability to knock out the autoreactive B-cell.

    • After that a Safety validation will be done to ensure the safety of the approach as a whole by testing:

      • Syn-notch: Co-culturing SUPER MSCs with non-targeted B-cells validates that it will not be activated unless it is the target one.
      • Exosomal receptor By adding exosomes to non-target B-cells, we will measure whether the exosomes will enter the non-target B-cells or not.
      • Safety switch We will add RFP just before the sequence and GFP just after the sequence. If the target cell is detected, it will express both colors (red and green). If it is not the target cell, it will express only red color.

    Finally, we will check the overall effect of the SUPER MSCs on the targeted B-cells.

    In vivo validation by animal model:

    We will address The Animal Model Qualification Program(AMQP) which provides a basic platform for collaboration with the FDA to facilitate animal model development. The qualification from the AMQP indicates that the FDA has concluded that the animal model can reliably produce a disease that corresponds to that in human beings. Furthermore, it indicates that the FDA has accepted the animal’s context of use which is the description of the use of the animal in the process. Rheumatoid arthritis animal models can be divided into two types: induced and spontaneous. We can use collagen-antibodies-induced mouse arthritis as it is considered the gold standard in rheumatoid arthritis trials. Arthritis needs to be induced through immunization against type 2 collagen and appears after 3-4 weeks after immunization. We can increase the severity by injecting lipopolysaccharides systemically. Also, we can use human TNF-alpha transgenic mice that develop arthritis spontaneously. Similarly, crossing autoantibodies against glucose-6-phosphate(K/BxN mice) leads to the appearance of arthritis spontaneously. Severe combined immunodeficiency (SCID)mice are considered humanized models of rheumatoid arthritis as they have been used to investigate human tissues in autoimmune diseases. The second step is to validate that these mice are suffering from arthritis and the progress after the injection of our therapy. Several experiments are made to assess the pain in mice suffering from arthritis. For example, pain-evoked behaviors, functional MRI, grimace scales, pain-suppressed behaviors, and operant conditioning and behavioral avoidance. Also, we have to follow up the inflammatory marker( ESR, C-reactive protein) and anti-CCP titer before and after the introduction of our new therapy.

    Clinical trial

    According to Food and Drugs Administration (FDA), (6) the pre-clinical research answers the basic questions about the drug safety, but it isn’t enough to start selling the drug in the market. We should go through clinical trial phase to validate the safety and the efficacy of the new drug. The clinical trial or research refers to the study of the drug's effect on the people to discover the possible and unpredictable interactions for the drug with the human body. Before starting our clinical trial, we should apply for investigational new drug application for FDA which includes:

    • Animal study data and toxicity (side effects that cause great harm) data.

    • Manufacturing information.

    • Clinical protocols (study plans) for studies to be conducted.

    • Data from any prior human research.

    • Information about the investigator.

    After the application, we will conduct to the clinical trial phases that are composed of 4 phases. Each phase will contain different number of patients that are appropriate for the information needed for each stage:

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    Fig 10. Shows the four clinical trial phases.

    Market authorization

    Federal Food, Drug, and Cosmetic Act (FD&C Act), and the regulations in Title 21- Code of Federal Regulations (21 CFR) Parts 1-58, 800-1299(7), settled laws to regulate medical devices marketed in the United States. The safety and efficacy of the medical device are the main determinant of the regulatory control and the marketing pathway of the device. To bring SUPER-MSCs to the United States market there are 4 steps to be followed:

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    Fig 11. Display market authorization steps.

    Step One: Classify Your Device and Understand Applicable Controls

    This stage includes 2 processes. Firstly, we should determine that our product is defined as a medical device by law in the section 201(h) of the Federal Food, Drug and Cosmetic. Then, we need to classify our product into one of 3 categories based on the degree of risk it has. According to law in the section 201(h) of the Federal Food, Drug and Cosmetic, SUPER-MSCs considered as a medical device. It is considered class III according to Product Classification Database which is the highest risk class in the classification.

    Step Two: Select and Prepare the Correct Premarket Submission

    There are different types of premarket submission according to the class of the product in step one. SUPER-MSC is a new drug classified to class III which has the highest risk. We need to apply by “Request for Evaluation of Automatic Class III Designation" under 513(f)(2) De Novo request” and “Premarket Approval”.

    Step Three: Prepare the Appropriate Information for the Premarket Submission:

    • Medical Device User Fees.

    • Small Business Determination (SBD) Program.

    • eCopy

    • eSTAR

    • Administrative Review.

    • Interactive Review.

    Step Four: Comply with Applicable Regulatory Controls, Including Establishment Registration and Device Listing:

    Real World Implementation

    Rheumatoid arthritis has an overall prevalence of 2.5% in South Africa with about 0.3% in Egypt. Egypt is a developing country that needs the help and power of the independent youth age group. As stated previously, RA causes deformity, in the long term, especially in hand joints which can interrupt the development process. This is why Egypt needs our project and needs to imply the concept of stem cell-based therapy generally. Firstly, we need to make stem cells bank and start taking samples from each person to have his own stem cells. Later on, we may add a new department for engineered stem cells. Engineered Mesenchymal stem cells require special requirements to be provided as a treatment:

    • Special requirements to be synthesized (cell source, media, temperature, technicians, biological parts).

    • Hardly stored.

    • Synthesized in response.

    Furthermore, we need to raise people’s awareness of the symptoms and signs of rheumatoid arthritis through fliers, counseling, advertisements, and brochures in primary health care. Moreover, people need to improve their fitness, nutrition, bad habits, and ergonomics. In tertiary health care and specialized centers, our therapy will be provided as a specific treatment for rheumatoid arthritis patients. Through the help of the Egyptian Ministry of Health and WHO, we can implement our project more accurately locally and globally, respectively.

    Routes of administration

    There are 2 methods of administration of MSCs: Intravenous and intraarterial. The most common method used in stem cell therapy is intravenous because it is easier with minimum procedural side effects. However, it could reduce treatment efficacy as most cells are trapped within small capillaries of various organs. On the other hand, the intraarterial route needs more skillful hands because it is hard to perform and record a lot of procedural side effects and injuries. Despite that, it displays higher efficacy in the treatment because the cells are not trapped in small capillaries. We suggest using the intraarterial route to reach the highest efficacy of the therapeutic platform.

    End users

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    Fig 12. Shows our targeted end users.

    Our target is to give rheumatoid arthritis patients a better life with less side effects from the old treatment modalities. So, SUPER-MSC should be used by professional team consisting of rheumatology consultant to confirm the diagnosis, immunology doctor to confirm the type of auto-antibodies affecting the patient, and biotechnologist which will take the responsibility of designing and manufacturing matching SUPER-MSC. Also, supervision of well trained doctors is needed to avoid any possible side effect caused by misuse. In addition, the SUPER-MSC will not be available in all hospitals because stem cells need special labs and conditions with low contamination to avoid any loss in the cells or decrease of its quality. Thus, the hospital must contain a stem cell unit,trained staff who has the ability to deal with stem cells, and biological parts which will be used to make SUPER-MSC.

    Future plan

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    Fig 13. Displays our future plan to use regenerative function of SUPER-MSC

    To implement our project, we have a short term plan to validate SUPER MSCs and to take FDA approval by following the steps we mentioned before. Then, we settled on a long term plan to improve the outcome of our therapy and to make it more familiar in the medical community. After that, we want to improve the diagnosis of rheumatoid arthritis by developing a criteria based on a clinical symptom appearing on the patient, laboratory findings, and Radiological findings. We started this phase by developing a mobile application that can differentiate between rheumatoid arthritis and other types of arthritis. The results of this application must be supported by clinical and laboratory findings.Moreover, we want to improve our therapy to make it a platform for all auto-immune disorders.Also, we want to regenerate the damaged organ using SUPER-MSCs by exploring its regenerative ability.

    Challenges

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    Fig 14. Illustrates the expected challenges we might face in the future.

    To implement our therapeutic approach in a community, there are a lot of challenges to make implementation easier and increase the efficacy of the treatment. The challenges during early stages of SUPER-MSCs' usage were:

    • Firstly,the source of MSC was one of the most important challenges facing us to ensure that we have a sustainable source for it. Therefore, we were planning to start an awareness campaign for pregnant women to give them information about stem cells and its importance not only in our project but also in biomedical research in general. Then, we will ask them to donate the umbilical cord after delivery guaranteeing informed consent. The umbilical cord is considered a rich source for stem cells, but unfortunately it is a wasted treasure in a lot of developing countries.

    • Secondly, after finding a sustainable source for MSC, we need to keep them safe and give them the optimum care they need. So, we are planning to build a central stem cell center that will work as a distributor for the cities surrounding. Let us take Egypt as an example for this system. We divided Egypt into 3 main regions: North Egypt center will be in Alexandria because it is considered the largest city there and already has a lot of research centers and experts that can take the responsibility for such a huge project. The second region will be middle Egypt or Great Cairo and it's the most crowded area. It will take place in the New Administrative Capital of Egypt. So, it must be a special center that has the capability to give his services to all the country and work as a backup for other regions. The third region is south Egypt or Upper Egypt which is less crowded and is known to be a rural community. As a result, we need to make the center in Asyut city because it is one of the largest cities. Also, there is a great transportation network from all of Upper Egypt cities. By this, we are sure that we have the essential infrastructure for our project to start.

    • Thirdly, we need skillful hands after we found the source of stem cells and prepared the infrastructure we needed for the project. As it is known most of the medical doctors don’t have enough experiance in synthetic biology to handle this project alone. On the other hand, biotechnologists don’t have a clinical background about rheumatoid arthritis. Therefore, we need to gather a team composed of two medical doctors and biotechnologist. The doctors will be from different departments: rheumatology and immunology departments to cover all the project aspects. All the team will be trained on the project to reduce the human mistakes.

    References

    • Usenbo A, Kramer V, Young T, Musekiwa A. Prevalence of Arthritis in Africa: A Systematic Review and Meta-Analysis. PLoS One. 2015 Aug 4;10(8):e0133858. doi: 10.1371/journal.pone.0133858. PMID: 26241756; PMCID: PMC4524637.
    • Cojocaru M, Cojocaru IM, Silosi I, Vrabie CD, Tanasescu R. Extra-articular Manifestations in Rheumatoid Arthritis. Maedica (Bucur). 2010 Dec;5(4):286-91. PMID: 21977172; PMCID: PMC3152850.
    • https://acrjournals.onlinelibrary.wiley.com
      /doi/10.1002/acr.24596
    • Yasir M, Goyal A, Sonthalia S. Corticosteroid Adverse Effects. 2023 Jul 3. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan–. PMID: 30285357.
    • Song N, Scholtemeijer M, Shah K. Mesenchymal Stem Cell Immunomodulation: Mechanisms and Therapeutic Potential. Trends Pharmacol Sci. 2020 Sep;41(9):653-664. doi: 10.1016/j.tips.2020.06.009. Epub 2020 Jul 22. PMID: 32709406; PMCID: PMC7751844.
    • https://www.fda.gov/patients/drug-development-process/step-3-clinical-research
    • https://www.fda.gov/medical-devices/device-advice-comprehensive-regulatory-assistance/how-study-and-market-your-device