Proof of concept

Expert validation

MSC

Dr. Yasser A. Aldhamen MSc, PhD, MBA: Associate Professor at Michigan State University Deputy Director of Research in the Faculty of Medicine at Michigan State University

Dr Yasser explained that

• MSCs have been extensively researched due to their documented inflammatory properties. Their ability to regulate the behavior of cells involved in responses positions them as a viable candidate for projects in this field.

• Promising results have shown that MSCs play a role in influencing the function of cells such as macrophages, dendritic cells, T cells, FLS and B cells. By controlling the activity of these cells MSCs can help manage responses and potentially alleviate symptoms associated with autoimmune diseases.

• Furthermore, MSCs are acknowledged for their effects which aid in restoring balance among individuals with autoimmune disorders. These remarkable cells possess the capacity to suppress responses and foster the production of regulatory cells thus enhancing overall immune tolerance.

• Additionally, another advantage of MSCs lies in their potential to facilitate tissue repair and regeneration. Research has demonstrated that MSCs possess properties that contribute significantly to tissue healing. This quality makes them an appealing therapeutic option, for conditions characterized by tissue damage.

Exosomes

DR. VELIA SICILIANO Principal Investigator, Istituto Italiano di Tecnologia, IIT

By speaking with Dr. Velia Siciliano about the utilization of exosomes in intracellular communication. Dr. Siciliano provided insights into the role played by exosomes in transmitting signals to auto-reactive B cells. This approach has the potential to address the response of cells to MSCs and prevent side effects that could be caused by proliferating MSCs.

Exosomes are vesicles that are released by cells, including MSCs, and contain a diverse range of proteins, RNA, and DNA. MSCs utilize these exosomes to regulate immunity within the body. Can also employ them for targeted delivery of agents via endocytosis. In terms of delivering cargo to autoreactive B cells using exosomes, the receptor on the exosomal surface consists of Lamp 2b presenting a CCP1 antigen that is recognized by autoreactive B cells.

Dr. Siciliano validated the concept of using exosomes for targeted delivery of agents to B cells and provided further clarification on the process involved. This approach holds promise in enhancing immunomodulation effects leading to a prolonged impact on target B cells while mitigating any risks associated with proliferating MSCs.

SYN-Notch

Dr. Mohamed El Saadawy Expert in research and laboratory work, professor in the Biochemistry at AFCM.

We discussed with Dr. Mohamed El Saadawy regarding his input on the idea of utilizing the Syn Notch receptor for laboratory experiments. We explained that this receptor is situated on the surface of cells and can be activated by either activity or contact between cells. Once activated it sends a signal within the cell through its transmembrane domain, which then triggers the release of transcription factors initiating a chain reaction in gene expression. The objective of this system is to present the CCp1 antigen on the surface of Msc cells, which can be recognized by autoreactive B cells. This signal is subsequently transmitted into Msc cells to generate exosomes containing cargo.

Dr. El Saadawy expressed support for implementing the Syn Notch receptor in laboratory work. He provided us with explanations and valuable insights into the process addressing any queries we had.

Safety Switch

Dr. Marwa Ali Professor of Biochemistry at the Armed Forces College of Medicine

She was really impressed with our approach to ensuring safety in the CRISPR Cas system. Being a professor of Biochemistry and a doctor herself she completely understands how crucial safety is in any procedure. She mentioned that our efforts in developing a safety switch are truly praiseworthy.

The DART VADAR switch you've created is a game changer in the field of CRISPR Cas technology. Its ability to detect and amplify RNA triggers using ADAR is truly remarkable. The fact that it ensures controlled translation based on the availability of ACPAs mRNA in auto-reactive B cells shows how thorough our team has been in guaranteeing maximum levels of safety.

She was particularly impressed with how selective and specific our design's targeting cells while minimizing off-target effects. Overall she believes that our work on the safety switch for the CRISPR Cas system makes a contribution to medicine. She fully supports our approach. She was excited to witness its impact on the medical community.

Modeling validation

The modeling part describes the binding kinetics between CCP1 of syn-notch receptor and BCR, In order to choose CCP1 as an external domain for syn-notch receptor we compared it with CILP and CV parts through their docking score for dissociation to BCR that happened after binding, So they prove that CCP1 is the most fitting part that describes that relation and also from the following graphs.

• CILP of syn-notch receptors shows a low rate of dissociation after binding to BCR as shown in graph (1) so it is not preferable to be used.

  

• CV of syn-notch receptors shows a high rate of dissociation after binding to BCR as shown in graph (2) so it is not preferable to be used.

  

• CCP1 of syn-notch receptors do not show a dissociation rate after binding to BCR as shown in graph (3) so it is the most preferable one to be used.

  

Also, the modeling proves that the activity of the internal domain (ZF21.16 VP64) of the syn-notch receptor is the most fitting part for the production of exosomes with acceptable concentration through compassion with different types of internal domains of other receptors. In addition, it is the most modular form of other receptors to produce a high concentration of our engineered exosomes.

• The signal from the internal domain of chimeric antigen receptors (CAR) can not induce exosome production as needed as shown in graph (4) because their internal domain can not be modified.

  

• The signal from the internal domain of receptors Activated Solely by Synthetic Ligands (RASSLs) can not induce exosomes production as needed as shown in graph (5) because their signals activate a lot of pathways so they are not specific for our circuit to produce exosomes as expected.

  

• The signal from the internal domain of antibody scaffold receptors (third generation of CAR) can not induce exosome production as needed as shown in graph (6) because their internal domain can not be modified.

  

• The signal from the internal domain of syn-notch receptors is the most acceptable one for exosome production as needed as shown in graph (7) because their internal domain can be modified to fit out expectations for exosome production.

  

Molecular dynamics:

To validate the ability of our SUPER cell to specifically identify our target auto-reactive B-cells that secrete the Citrullinated Peptide Antibodies (ACPAs). We evaluated the interaction between auto-reactive BCR and different designs of our syn-notch receptor to select the most efficient possible candidate that enables our MSCs to efficiently sense the presence of the target auto-reactive cells while sparing the body’s normal immune cells.

That’s why we performed docking and molecular dynamics simulations between the variable region of the auto-reactive B-cell receptor that resembles the variable region of ACPAs synthesized from these cells and some of its targets to select the optimum protein in terms of structural stability and binding affinity in order to improve the specificity of our engineered SUPER cells.

Molecular docking simulations of different designs of our syn notch receptor:

The docking simulation was conducted using the HDOCK server developed by Huang lab. These simulations test the structural stability and binding affinity between the target auto-reactive B-cells with three different proteins that represent the extracellular domain of three different designs of our syn notch receptor.

These proteins are:

• The citrullinated vimentin (CV).

• Cartilage intermediate layer protein (CILP).

• The Cyclic Citrullinated Peptide (CCP1).

The docking results illustrate that the most stable binding between our three designs of the syn notch receptor and the auto-reactive BCR was conducted with CCP1, with a score of (-298.80), followed by CLIP with a score of (-242.04), and CV was the lowest with a score of (-160.74).

Molecular dynamic simulations :

MD was conducted using the AMBER molecular dynamic packages on Google Colab in order to simulate the previously mentioned complexes to assess the stability of their molecular structures in a perturbed environment, simulating what they would face within our engineered actual cells.

From the previous three candidates, CCP1 simulations showed the most stable structure and the best flexibility as the RootMean Square Deviation (RMSD) was between 1.5-2.5 Å, which means minimal deviation between the atoms of CCP1 and the target BCR which reflects the stability of the formed complex. The value of the Root Mean Square Fluctuation (RMSF) also ranged between 2-3 Å, which indicates minimal fluctuation in the position of each residue forming the structure of each protein.

CCP1-target BCR complex molecular dynamics:

CV-target BCR complex molecular dynamics:

CLIP-target BCR complex molecular dynamics:

To prove the concept of our approach we had to co-culture our engineered cells with auto-reactive B-cells that secrete ACPA but we had a problem with the availability of the auto-reactive B-cell due to the complexity of their isolation process and lack of instruments, so we had to modify our design to fit with the available material that's why we have changed the extracellular domain CCP1 sensing the auto-reactive B-cells to anti CD-19 as we found that CD19 is over expressed on the surface of B-cell specifically and other immune cells that allow us to validate our approach experimentally. so we decided to test the validity of our design before proceeding to the next step.

That’s why we conducted docking and molecular modeling for the CD19 and anti-CD19 to measure the binding between them in addition to validating the stability of their structures in a physiological system. The docking results between their structures was (-311..10), and when their structures were submitted for molecular dynamics simulation, their RMSD reached 5 Å as well as the values of their RMSF.

Lap validation :

Our biological circuit is designed to be assembled into three pcDNA3(-) eukaryotic expression vectors that are labeled with different reporter genes to be transfected into the WI-38 cell line through a poly transfection technique based on lipofectamine.

The transfection and the integrity of our biological circuits would be validated through flow cytometry that purifies the appropriate cells that contain our three plasmids and express their three different reporter genes thus the expression of our biological parts would be validated as a whole.

Our plan:

We intended to prove the concept of our design through two major steps that validate our design's effectiveness and specificity.

• structural validation

• functional validation

Structural validation:

The first step is structural validation of our biological parts through tagging and integrating different reporter genes that reflect the successful expression of our biological parts structurally to be presented in the intended site, desired amount, and at the right time.

Syn notch structural validation:

We intend to validate the constitutive expression of our synthetic notch receptor by tagging the presented receptor with a His tag peptide to be detected by fluorescence-labelled antibodies secondary that binds to his tag primary antibodies through a sandwich technique to visualise the presentation of our synthetic receptor on the surface of the cells by fluorescence microscope.

Exosomal receptor structural validation:

This receptor would be validated by the same technique mentioned before with Syn notch receptor as it’s labeled with flag tag peptide that would reflect the expression of the receptor on the exosomal surface by fluorescence-labeled secondary antibodies that bind to flag tag primary antibodies through sandwich technique.

Therapeutic cargo structural validation:

As the expression of our cargo is conditioned by the activation of our synthetic receptor by binding to its target cell. This step would require co-culturing of our engineered WI-38 that expresses our anti-CD19 syn notch receptor with Epstien-Barr lymphoblastoid B-cells that replace auto-reactive B-cells in the design to prove our concept.

This step is considered functional validation of the receptor to prove its ability to control the expression of the therapeutic cargo and structural validation of our therapeutic cargo.

Therefore, we had to implement a control line by co-culturing our engineered WI-38 cell line with other cells that present limited copies of CD19 as a natural killer thus we would compare the levels of fluorescence protein expressed in each plate and quantify the amount of our expressed cargo in the form of mRNA through northern blotting

Aiding circuits (CX43-Booster genes) structural validation:

These parts validation was intended to be done through reporter genes located upstream to their sequences that ensure the integrity of our circuit as well as the functionality of our promoters.

Functional validation:

The second step is functional validation of our biological components to ensure their ability to perform what was intended to be done and characterize their action and performance.

Synthetic notch receptor functional validation: As this part validation was mentioned before the performance of the synthetic receptor would be indicated through a reporter gene (GFP) expression measurement that reflects the ability of our transcription module VP64 within the intra-cellular domain to conditionally induce transcription of our therapeutic cargo(Cas12k) through ZF21.16 promoter.

Booster genes functional validation: We intended to validate the functionality of our booster genes (SDC4, STEAP3, NadB) and their ability to enhance or increase the default levels of exosomal secretion by characterizing the size distribution and concentration of exosomes after isolation by centrifugation of the synthesized exosomes from multiple areas within the sample. To measure the concentration of exosomes in different areas to determine the average level of exosomal secretion by Nanoparticle Tracking Analysis(NTA) and characterize the shape of the exosomes by Transmission Electron Microscopy (TEM).

Tissue-specific switch functional validation: To validate the performance of the DART-V-ADAR tissue-specific switch we intended to co-culture our engineered exosomes that contain our therapeutic cargo with lymphoblastoid B-cells in order to transfer switch-Cas12k RNA transcripts to these cells in order to mediate their apoptosis. Therefore, we have changed the sensor sequence to be complementary with the B-cell activating factor receptor mRNA as a result the hybridization reaction between the sensor and BAFF-R mRNA mediates (UAG) stop codon deamination into (UIG) leading to the expression of the downstream reporter gene (RFP) that would be quantified through flow cytometry.

Cas12k-g(BAFF-R) functional validation: To assess the functionality of our CRISPR-Cas12k system which is designed to knock down the BAFF-R gene we would introduce our engineered exosomes to lymphoblastoid B-cells to impair its survival and modification by affecting the expression of BAFF-R, Therefore we would validate our therapeutic cargo by cell surface staining technique to analyze membrane presentation of the BAFF-R by using a Gillios Flow Cytometer.

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