Project Description

Disease Background:

Rheumatoid Arthritis (RA) is a chronic inflammatory autoimmune condition that mainly targets the lining of a patient’s synovial joints, mainly affecting the small joints of the hand and wrist, but it can also affect any other joint in the body. It is mainly characterized by joint pain, redness, swelling, and stiffness, which can develop insidiously over the course of weeks or even months. The destructive potential of RA can result in irreversible joint deformities that can prevent an individual from carrying out their work and in turn have a great impact on their lives.

The condition can also affect multiple body systems; including:

• The eyes: can result in prominent dryness that can lead up to corneal ulceration.

• Lungs: increase the risk of developing lung fibrosis and fluid build-up between the lungs and their surrounding pleura.

• Cardiovascular system: result in premature atherosclerosis that can increase the risk of having a heart attack.

Etiology and risk factors for developing RA:

The etiology of RA is multifactorial and depends on the interaction between various risk factors; including the genetic background, environment, and socioeconomic status.

• Genetic Predisposition:
  It has been shown that 40-65% of seropositive rheumatoid patients are more likely to pass the condition to future generations. The development of RA has also been associated with a number of Human Leukocyte Antigen (HLA-DRB1) alleles; including (HLA-DRβ1*04), (HLA-DRβ1*01), and (HLA-DRβ1*10). All of these alleles contain a conserved sequence known as the Shared Epitope (SE), which is a 5-amino acid sequence motif that has been correlated with increased susceptibility of developing a more severe form of RA.

• Environmental factors and socioeconomic status:
  Cigarette smoking has also been identified as one of the strongest environmental factors to be correlated with the occurrence of RA. It has been shown that the interaction between tobacco and the shared epitope is associated with an increase in the risk of RA.

Other risk factors include the exposure of certain fumes and gasses, which are commonly found in construction and industrial work. This includes the inhalation of metal fumes, silica dust, textile dust, and asbestos. These findings increased the probability of considering RA among male workers to be an occupational disease.

Epidemiology background and impact of RA:

In 2019, the World Health Organization (WHO) has estimated that over 18 million people were living with RA all around the world, which is double the number of RA cases since the year 1990. According to the Global Burden of Disease (GBD) estimations, there has been a marked increase in the prevalence and incidence of RA worldwide.

Globally, there has been a marked worldwide increase in the incidence of RA cases each year. The number of new RA cases has nearly doubled in Egypt and the Middle East and North Africa (MENA) region between the years 1990 and 2019.

In the last 3 decades, the prevalence of RA has nearly tripled in the MENA region, reaching nearly 650000 cases in 2019 when compared to the number of cases in 1990.

Egypt has also shown an increasing trend in terms of prevalence and incidence of RA cases.

Another important parameter to be considered is the calculated Disability-adjusted life-years (DALYs), which is used to calculate the number of years that patients lose due to their condition, either due to premature mortality or due to living with the disability itself. The DALYs have also shown a notable increase in the number of years that RA patients lose due to their disability. The overall calculated DALYs for RA patients in Egypt was estimated to be more than 12000 years, which is triple the number it was in 1990.

All of the aforementioned statistics indicate that the burden of RA, especially in the MENA region, has shown a steady increase between 1990 and 2019. This indicates the necessity of providing urgent interventions and solutions that can control this massive expansion of RA, not only in our region, but all around the world.

Pathophysiology of RA:

As previously mentioned, the development of RA depends on the interaction between various environmental and socioeconomic factors in the presence of a genetically-predisposed individual . This makes the body’s immune system unable to tolerate self-proteins that contain the citrulline residue. The process of forming the citrulline residue has been found to be overly active at sites of joint swelling and inflammation that are associated with activity of RA. The citrullination process is mediated by an enzyme called peptidyl arginine deiminase (PAD), which turns arginine residues into citrulline. Some polymorphisms in the gene coding for this enzyme have also been associated with the development of RA, including the PADI2 and PADI4.

The pathophysiology of the condition depends mainly on the presence of auto-antibodies against citrullinated peptides in the body.

Many of these antibodies have been identified in the literature and they include Rheumatoid Factor (RF), anti-citrullinated peptide antibody (ACPA), anti-carbamylated protein antibodies (anti-CarP antibodies), Anti-PAD4 Antibodies, and Anti-Modified Protein Antibodies. However, RF and ACPA are the ones which are mainly implicated in the pathogenesis of the disease.

Numerous cytokines are also involved in the pathogenesis of RA as they are produced in abnormally large amounts due to recruitment and migration of different inflammatory cells into the joint cavities.

The process of swelling, inflammation and joint destruction occurs as a result of the interaction between components of the body’s innate and adaptive immunity, in addition to being influenced by the produced cytokines.

The immune cascade of RA pathogenesis and progressive tissue destruction occurs as follows:

• The abnormally citrullinated peptides are phagocytosed by macrophages and dendritic cells present in the lining of joints, in order to be conjugated with MHC-II and presented to Helper-T cells (Th).

• The activation of Th cells requires two signals: the first one is the antigen presentation done by antigen-presenting cells (APCs) like macrophages and dendritic cells. And the second one is a costimulatory signal produced by the interaction between the cell surface protein CD80/86 on APCs and the CD28 protein of Th.

• Meanwhile, a massive amount of cytokines are produced to suppress the production of Regulatory-T cells (Tregs) and increase the activity of APCs; thus increasing the rate of the inflammatory destructive process. These cytokines involve IL-1β, IL-6, and TNF-α.

• B-cells play a vital role in the pathogenesis of RA since their contribution to the autoimmune process is mediated through many different pathways. They can act as APCs that can activate Th, and they can also contribute to the synthesis and release of proinflammatory cytokines. However, their pivotal role is represented in their ability to differentiate into plasma cells and produce auto-antibodies. The most prominent of these antibodies is the RF and ACPA, which bind to their antigen forming immune complexes that can further complicate the inflammatory destructive reaction by activating the complement system.

Available treatment options:

Conventional pharmacological options include corticosteroids and other steroid-sparing immunosuppressants such as methotrexate (MTX), azathioprine, cyclosporin, and hydroxychloroquine. These drugs can be used as anti-inflammatory agents that help decrease the impact of the destructive inflammatory reaction. However, these drugs result in non-selective overall immunosuppression, which can make the patient more susceptible to opportunistic infections. In addition, they are also associated with a number of dangerous side effects such as affecting the liver, kidneys, and even bone marrow.

Another class of drugs include biological drugs, which are designed to target specific inflammatory cells, cellular interactions, or even cytokines along the immune response cascade of RA. Although the efficacy of these drugs is much better than other therapeutic options, they are very expensive and not steadily available.

The most important risk associated with all of the aforementioned drugs is that they require lifelong administration since RA is an autoimmune disease, which also means lifelong exposure to the side effects associated with these drugs.

That’s why we had to come up with a different treatment option in order to avoid all of these risks and improve the quality of life of our patients.

Introducing our therapeutic approach:

This year, we’re looking to use the powerful potential of Mesenchymal Stem Cells (MSCs) to design an innovative therapeutic solution for RA. Our ultimate goal is to design a modular, controllable system that can control the progression of the condition, without having to deal with other side effects that can be associated with the usage of immunosuppressive drugs.

Introducing our SUPER-Cell: Smart, Universal, Pleomorphic,Endogenous, Regulator of immunity.

Our approach is based on engineering MSCs to:

• Specifically identify and bind to auto-reactive B-cells that are responsible for the production of the ACPA.

• After recognition, release our engineered exosomes occurs to target the auto-reactive B-cells and deliver our therapeutic genetic cargo in order to destroy these cells and decrease their release of ACPA.

• In addition, MSCs can play a vital role in suppressing the inflammatory process associated with RA. This is achieved by down-regulating the release of pro-inflammatory cytokines, while up-regulating the release of anti-inflammatory cytokines.

• This would eventually decrease the progression of the disease, while sparing the normal function of the immune system against harmful microbes and microorganisms.

Our ultimate goal is to turn our approach into a modular therapeutic platform that can be engineered to target and manage many other autoimmune conditions, in order to help as many patients as we possibly can.

Our design:

The design of our project is mainly structured on 5 pillars:

1) Engineered MSCs:

Mesenchymal Stem Cells have been reported to be widely used in cell-based therapies. A number of studies have discussed their therapeutic and regenerative potential in treating different conditions such as bone, cartilage, or even some neurological conditions. They are also known for their ability to regulate the overactive response of the immune system in autoimmune diseases.

They have also been used in clinical research targeting RA. They have been shown to ameliorate the destructive impact of RA by regulating the activity of T-cells. This is achieved by suppressing the differentiation of Helper-T cells into Th17 cells, which play an important role in the inflammatory activity of RA. In addition, MSCs are responsible for the up-regulation of Regulatory-T cells (Tregs), which help in controlling the magnitude of the immune response.

Furthermore, it has been shown that MSCs decrease the production of pro-inflammatory cytokines that are implicated in the pathogenesis of RA, such as IL-1β, IL-6, and TNF-α. This is done in conjunction with increasing the release of anti-inflammatory cytokines like IL-10, which contribute to decreasing the aggressive inflammatory response, while promoting tissue regeneration.

MSCs have also been shown to modulate the humoral immune response, which mainly depends on the production of antibodies by activated B-cells. The effect of MSCs-based therapy was also tested during a study that was conducted by Gowhari et al. in 2020. This study has shown that MSCs also affect the activity of ACPA-producing B-cells by decreasing the concentration of the B-cell Activating Factor Receptor (BAFF-R), which is essential for B-cell survival and differentiation. The coding gene for this receptor is also targeted by our CRISPR/Cas12k genetic circuit (which is discussed later on in the CRISPR/Cas section).

Despite the numerous advantages of using MSCs in cell-based therapies, they have been associated with some undesirable adverse events. Some papers have discussed the tumorigenic probability of these cells in case of increased unchecked proliferation. That’s why, we’ve implemented a number of safety measures in order to keep the activity of our MSCs under control. We’ve integrated an iCaspase 9 Suicide System (IC9) into the design of our engineered MSCs. This system can enable external control of the activity of our cells by injecting an element called the Chemical Inducer of Dimerization (CID). This element activates the IC9 system within these cells, which induces apoptosis of the engineered MSCs and terminates their effects. (Kindly refer to the Safety Page for more information)

Our MSCs have been engineered to include 3 distinct plasmids, each has its own unique function:

• The first plasmid is designed to express our synthetic ‘Syn-Notch’ receptor.

• The second plasmid includes the loading system of our therapeutic cargo into the exosomal delivery system, in addition to including booster genes that enhance the release of exosomes from the MSCs.

• The third and final plasmid encodes for our CRISPR/Cas12k cargo, which is intended to be loaded into our exosomes in case our MSCs identify any auto-reactive B-cells.

The first plasmid is designed to be constantly active to produce the Syn-Notch receptor that is responsible for the recognition process. As for the other 2 plasmids, their expression is controlled by the activity of the Syn-Notch. If binding the Syn-Notch and auto-reactive cells occurs, the receptor internal domain activates the other 2 plasmid to initiate the release of our exosomal delivery system to target the auto-reactive cells with its therapeutic cargo.

2) Our synthetic ‘Syn-Notch’ receptor:

We’ve designed our SUPER-Cells to express a synthetic receptor called the ‘Syn-Notch’. This receptor is composed of 3 distinct domains: an external domain, a transcellular domain, and an internal domain.

The receptor’s external domain is engineered to express the Cyclic Citrullinated Peptide (CCP1) antigen and is complementary to the ACPA receptor which is specifically present on the surface of auto-reactive B-cells in RA. The CCP1 is presented extracellularly using a CD8-α molecule, that ensure its stability. This allows our MSCs to specifically target the auto-reactive B-cells while sparing other normal B-cells, which also acts as one of our safety measures to ensure selective targeting of the autoimmune cells.

The transcellular domain of our receptor is called the ‘Mouse Notch’. This part is responsible for maintaining the structural stability of our receptor, as well as transmitting the signal from the receptor’s external domain into its internal domain, in case the binding occurs with an auto-reactive B-cell.

As for our receptor’s internal domain, it is composed of a transcriptional activating factor called ‘ZF21.16-VP64’. This transcription factor regulates the action of our genetic circuit.

When our MSCs identify the auto-reactive B-cells through the interaction between the external domain of our Syn-Notch receptor and the ACPA receptor of B-cells, a signal is transmitted through the transcellular part of our receptor, until it reaches the receptor’s internal domain. This results in the dissociation of the the internal ‘ZF21.16-VP64’ from the receptor in order to activate our circuits, to initiate the release of our exosomal delivery system, which is designed to target the auto-reactive cells. (will be discussed in the Exosomal Delivery System section)

3) Exosomal Delivery System:

Another important feature of MSCs is their ability to release extracellular vesicles called ‘Exosomes’. These vesicles have been documented in the literature for their extensive and wide range of applications as delivery vehicles. They can be used to selectively deliver drugs, microRNAs, genetic circuits, or even complex proteins. That’s why we’ve decided to make use of this unique property of MSCs, in order to use the potential of the produced exosomes to deliver our genetic cargo into the autoimmune cells.

Once the recognition process between our MSCs and the auto-reactive B-cells is done, the Syn-Notch’s internal domain is proteolysed into a transcription factor that activates the transcription of our CRISPR/Cas12k cargo in order to be loaded onto our exosomes. We included a non-coding RNA motif called the ‘C/D Box’ in the desing of our cargo , to guide it into the exosomes.

The loading process of the exosomes occurs as follows:

• Our exosomes are designed to produce a CD63 surface antigen, whose internal aspect involves three copies of the ribosomal protein ‘L7Ae’, which contains another C/D Box that is compatible with the C/D Box of our cargo.

• This allows our cargo to be directly loaded into our exosomes, which are then released to target the auto-reactive B-cells and deliver its cargo. The released exosomes are also expressing the CCP1 antigen in conjugation with a glycoprotein called the LAMP-2B, which allows for specific targeting of the auto-reactive cells.

4) CRISPR/Cas12k Cargo:

After their release, our exosomes start targeting the auto-reactive B-cells in order to deliver their contents. Our cargo is composed of the CRISPR/Cas12k system (transcriped in the RNA form), in addition to a guide RNA that directs it towards a specific gene in the B-cells called the B-cell activating factor receptor (BAFF-R) gene. This gene encodes for an activating factor that is essential for the survival and differentiation of B-cells.

When the exosomes bind with an auto-reactive B-cell, they are endocytosed into the B-cell so as to release their cargo. The Cas12k system is then guided towards the (BAFF-R) gene, resulting in knocking it out and initiating apoptosis of the auto-reactive cells.

We’ve chosen the Cas12k system because of its small size which can be tolerated by our exosomes. In addition, this class of CRISPR/Cas targets double-stranded DNA (dsDNA), which also serves the purpose of our approach to target the BAFF-R gene of the auto-reactive B-cells.

5) DART/VADAR Safety Switch:

In order to ensure the safety and control of our therapeutic system, we’ve incorporated a tissue-specific safety switch into the design of our genetic cargo. This switch is designed to prevent the expression of our CRISPR/Cas12k unless it is present within the environment of auto-reactive B-cells.

The safety switch depends on a sensor called DART-VADAR (Detection and Amplification of RNA Triggers via ADAR enzyme). The ADAR (Adenosine deaminase acting on RNA) component of the sensor is an enzyme that catalyzes the post-transcriptional conversion of Adenosine (A) into Inosine (I). The switch is designed to include the stop codon (UAG) and is positioned upstream to the coding sequence of the CRISPR/Cas12k. Finally, we’ve designed the switch to be complementary to the mRNA sequence of the ACPA, which is specifically found within the auto-reactive B-cells. The change in the sequence of the stop codon into (UIG) will expose the downstream CRISPR/Cas12k to the cell’s ribosomes for translation to guarantee that our system will not be activated even if our exosomes target other colonies of B-cells.

Our vision

We’ve chosen RA to be a proof-of-concept of the efficacy of our approach. But, we believe that it has the potential to be modulated into a universal therapeutic platform for many other autoimmune conditions. This would not only enable us to help RA patients, but also provide hope for many others who are suffering from the burden of autoimmune conditions, which affect nearly 10% of the global population.

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