Engineering Success

Introduction

To tackle one of the most widely spread autoimmune diseases in our region, our journey has gone through various phases of Design and Research-Build-Test-Learn. We started with a general study of the different autoimmune diseases prevalent in our society. After a lot of hard work and long hours of deep research, we came up with a long list of a variety of serious and widely spread diseases. On top of that list came Rheumatoid Arthritis (RA).

RA is one of the oldest yet the most common diseases in our society. Its unknown etiology and lack of terminal specific cure made it our undisputed target in 2023.

Therefore, we started working with one goal in mind, and that is to create an effective treatment for Rheumatoid Arthritis . On our adventure against this disease, we came across a lot of challenges that have only inspired us to keep going.

Here, we will take you on a quick tour through the different stages of our project.

Iteration 1:

Design and research:

Our literature search demonstrated that macrophages play an important role in the pathophysiology of Rheumatoid arthritis by inducing inflammation and increasing disease activity, which can also be one of the factors that can cause joint destruction.

They can also act as antigen-presenting cells, which can stimulate the activity of other inflammatory pathways, resulting in further tissue destruction.

Build

Our first iteration was to genetically engineer the macrophages to make them able to suppress the immune reaction. This was achieved by switching them from the M1 Inflammatory phenotype to the immunoregulatory phenotype M2, which regulates and suppresses other immune cells’ activity while promoting tissue regeneration, thus limiting the progression of the disease.

Test

Through a deeper search, we found that macrophages are not the only cells responsible for the damage caused by Rheumatoid Arthritis.

So the effect of the phenotypic switch won't be enough to significantly control the disease activity.

Learn

We wanted to have a broader look for other factors damaging the joints and corrupting the immune response, such as Fibroblasts-like synoviocytes (FLS), autoreactive B-cell antibodies, and T cytotoxic cells. So we decided to work on another approach targeting one of these main factors.

Iteration 2:

Design and research:

Our research proved that the pathophysiology of Rheumatoid arthritis is multifactorial, not just affected by one or two pathways; there are multiple factors affecting the disease pathophysiology, such as T-cells, B-cells, macrophages, and fibroblasts like synoviocytes (FLS), etc.

Build

Our second iteration was to modify one of the most commonly engineered cell-based therapies, which is T-cells. These modifications will take place through genetic engineering to make T-cells able to detect and suppress the immune reaction. This was achieved by targeting and destroying the auto-reactive B-cells that produce ACPA antibodies that induce inflammation and destruction, both local and systemic, thus limiting the progression of the disease.

Test

Through considerable exploration, we found that the auto-reactive B-cells are not the only cells responsible for the damage caused by Rheumatoid Arthritis.

On the other hand, using T-cell-based therapy results in ramping up the immune response. This means that T-cells could increase the inflammatory response and also suppress normal healthy cells, not only the auto-reactive B cells.

Therefore, this design of the therapeutic approach wouldn't reach the intended optimum results in terms of achieving best specificity and controlling disease activity, as well as having many undesirbale side effects.

Learn

We decided to look for other cells that can be used for cell-based therapy. But this time, we will look for cells that can regulate more than one pathway and -at the same time- have a few adverse effects on immunity. We made patient’s safety and drug’s specificity our top priority. Hence, we started looking for another approach that could control disease activity without significant unwanted effects on other normal healthy systems.

Iteration 3:

Design and research:

Our search was conducted to find a vector or a cell that has wide control over most of the factors responsible for the pathophysiology of rheumatoid arthritis. After going through many papers and consulting many experts, we finally decided to take advantage of Stem cells as an innovative therapeutic approach, specifically Mesenchymal Stem Cells (MSCs).

Build

Our third iteration was to modify MSCs; to make them directed specifically towards the site of interest. Therefore, we had to add an extracellular receptor, making MSCs able to detect the fibroblast-like synoviocyte (FLS). As a result, activated MSCs act by regulating autoimmune cells' function and suppressing their pathogenic effect on joints by secreting their built-in exosomes that regulate the FLS to decrease disease activity. Besides that, MSCs also have an amazing feature; they naturally contribute to the regeneration of damaged tissues in the joints.

Test

Despite all the great aspects of MSCs and their contribution to control the disease, we found that their activity remains non-specific and uncontrollable. In addition, there is no significant effect on the disease activity, as the FLS is not the only cell contributing to the pathophysiology of the disease. Our design also lacks specific targeting of the FLS, which reduces its potential as a great treatment option for RA.

Learn

We concluded that we need to increase the effectiveness and the specificity of our approach. Furthermore, we wanted to target one of the main cells responsible for the pathophysiology of the disease , rather than just depending on the great built-in functions of the MSCs.

Iteration 4:

Design and research:

Through a deeper search and long brainstorming sessions, the idea of using exosomes as a vector synthesized by MSCs came to light. Exosomes are a class of cell-derived extracellular vesicles of endosomal origin; they also play an important role in intercellular communication.

Build

In the fourth iteration, we wanted to isolate the exosomes first from MSCs and modify these exosomes to carry the apoptotic signal called BID to initiate the apoptotic pathways in the T-cells and B-cells. We also added a targeting receptor on the exosomes to ensure that the cargo reaches and suppresses the auto-immune cells.

Test

The application of this design proved that the BID signal carried by Exosomes is still not specific enough to the targeted auto-immune cells. In fact, there are more than 200 normal peptides targeting various cells, making them victims of apoptosis due to off-targeting of this signal. Also, exosomes have quite a short half life, they require multiple doses to achieve their purpose.

Learn

We found that our last design needs more modification in order to prevent the off-targeting effect on other normal immune cells caused by the multiple peptides on the surface of the exosomes. We also needed to focus on developing the BID apoptotic signal, making it more selective for the targeted B cells. This could be done by either substituting this signal with another one or genetically modifying this signal with safety switches.

Iteration 5:

Design and research:

After contacting Immunology & Rheumatology experts and meeting with Stem cell specialists, we learned that the auto-reactive B-cell is considered one of the main cells causing the pathogenesis of RA. Since the concentrations of B-cell produced ACPA titre correlate with the severity of the condition. We also found out that the inflammatory effects of ACPA are not just limited to joints but also reach out for other tissues resulting in severe damage.

Build

In our fifth iteration, we went back to working on MSCs. Hence, we started to modify the extracellular domain of the MSCs’ receptor to directly recognize auto-reactive B-cells. This was achieved through using one of the citrullinated antigens found in the literature and testing their binding affinity to our engineered receptor by simulating it through software tools.

Test

We found that the binding affinity of MSCs with autoreactive B-cells was not high enough, which causes the lack of specificity of this design. We also faced the same problem in the previous iteration where we depended mainly on the natural effects of MSCs in enhancing the immunity and regulating the inflammatory response.

Learn

We discovered that we need to make more modifications to our last design to increase the specificity by finding another ligand. We also need to integrate another system into our design making it more specific to the auto-reactive B-cell and regulating its activity. On another hand, we felt that we could benefit much more from the various potentials of MSCs by modifying them with genetic engineering.

Iteration 6:

Design and research:

During our search, we tried to simulate the binding between our receptor and its target to test the stability inside the MSCs. Therefore, the literature taught us that there are different types of synthetic receptors that can be used on MSCs with a modifiable extracellular domain. The type of receptors depend on the nature of the target which may be a cell or a molecule.

Build

Our sixth iteration was to modulate and choose a type of receptor suitable for our design, so we went through six types of receptors from GPCRs, CAR, CAAR, MESA, GEMs, and others. After prolonged research and various tests, we finally settled on the SYN-Notch receptor.

Test

We found that the SYN-Notch receptor was the most suitable for our design. This receptor is characterized by its ability to sense cells in the extracellular environment. Adding to that, its modular ability to regulate the extra and intra cellular domains. It is also considered a stable receptor thanks to its Mouse Notch Intermembrane domain.

Learn

After we settled on the type of receptor, we wanted to improve our approach by increasing the binding ability of the receptor and also modifying our genetic parts, both by using directed evolution. All along, we were eager to reach the best form of our design either by applying more modifications or merging different approaches or new designs together.

Iteration 7:

Design and research:

After our successful attempts to modify the receptor and genetic parts, we needed to settle on a better approach that could guide us to an optimum design. Therefore, we had the idea of merging our prior approaches together (MSCs and Exosomes), which led to the third integrated design in our project.

Build

Our seventh iteration was the integration of the prior approaches, MSCs and Exosome-based therapy. We developed MSCs capable of secreting exosomes containing a BID signal that initiates apoptosis within the autoreactive B-cell. In addition, we implemented booster genes in our circuit in order to increase the expression of exosomes from the MSCs.

Test

Our integrated approach has greatly improved our platform. As a result, our design can widely control and treat RA by regulating the multiple factors responsible for the pathogenesis.

Moreover, we modeled the kinetics of using booster genes to amplify the exosome production as shown in the following graph.

However, we still needed to make some modifications to our platform to assure the applicability of our therapeutic approach as an effective drug for RA.

Learn

We decided to modify the BID signal inside our exosomes in order to become more targeted, specific and stable. We were also keen on enhancing the signal’s ability to inhibit the auto-reactive B-cells.

Iteration 8:

Design and research:

After many unfortunate attempts to modify the BID signal of the exosomes, we came up with another idea of using another system instead of BID. This new system should be more specific and effective in inhibiting the auto-reactive B-cells.

Build

In our eighth iteration, we replaced the BID apoptotic signal as we developed exosomes with a CRISPR-Cas system that knocks down the B cell-activating-factor-receptor (BAFF-R) gene, which is an essential factor for B-cell survival.

Test

This design was considered the peak of our therapeutic journey as it has become much more effective and selective than its priors. However, we still need to assure the safety and feasibility of our treatment by sparing normal B cells from the silencing effect of the CRISPR-Cas system, while at the same time, suppressing the inflammatory cells implemented in the pathogenesis of RA.

Learn

We thought about adding a new modification into our circuit in order to reach a safer version of our integrated design. This modification would aim to regulate the disease activity and its progression while preventing the silencing effect of the CRISPR-Cas system on the normal B-cells.

Iteration 9:

Design and research:

To increase the safety of our approach, we decided to choose a new family of the CRISPR system and we also wanted to regulate the expression of our engineered exosomes. Hence, we decided to look for a method that can control the translation of the Cas9\g\mRNA ACPA system. As a result, we could regulate the binding of the Syn-Notch receptor with its targeted autoreactive B-cell.

Build

In our ninth iteration, we decided to choose the CRISPR-Cas12k system as our new CRISPR family and adding a new layers of safety to our approach. Therefore, we implemented the cre-recombinase system to the booster genes to control their expression. Secondly, we inserted a new tissue specific switch in our circuit called DART-V-ADAR (Detection and Amplification of RNA Triggers via ADAR). This switch will guarantee a conditioned translation for our system, based on the availability of ACPA’s mRNA in the targeted cells.

Test

Our design has reached a high level of specificity and selectivity with the Cas12k\g\mRNA ACPA system controlled by the tissue-specific switch. As a result, our platform can deplete the autoreactive B cells safely with a minimal off-targeting effect.

Thanks to the modeling, we found that booster genes added to our circuit would increase exosomes production 1.2 times the normal release in absence of booster genes.

Learn

We decided to adapt this last integrated approach and start our Wet Lab work as soon as possible.

Iteration 10:

Design and research:

Fortunately, the people responsible in the airport agreed to help us by speeding up the process of the delivery so we could receive our order in the shortest time possible .

Build

After more than one month from our order, we finally received our parts and materials. Hence, without any further delay, we directly arranged all the lab requirements and launched our wet lab phase. We started working on resuspension, amplification and digestion protocols and -at the same time- bacterial transformation for our vector.

Test

We tried to resuspend all the g-Blocks, amplify using PCR, and start the digestion protocol. We also began running the parts on gel to see if the digestion process would go well.

Learn

Unfortunately, we found that the concentration of all the DNA parts is too low to go through the ligation process. troubleshooting may be due to:

• Provided parts( inserts) were not ordered in cloning vectors that are known for easier restriction and ligation, while linear fragments have too many issues.

• Parts were provided in low concentration.

• Primers were not designed as recommended.

• It is better to do the assembly using Gibson Assembly , Overlap PCR or 3A assembly.

• The provided restriction enzymes are of low efficiency.

Iteration 11:

Design and research:

We reached out to some doctors and experts to help us improve and fix this issue. Therefore, they advised us to adopt another methods such as TA cloning method or increasing the PCR reaction.

Build

In this iteration, we ordered the new essential kits for this suggested approach in order to start implementing them in our design. However, we were running out of time as we had only a couple of weeks for the wiki freeze, so we decided to increas the PCR reaction untill recieving the kits .

Test

In order to use TA cloning, we had to make our order as fast as possible from the available resources and running PCR amplification with higher PCR reaction that result in increase the purified concentrations of DNA parts.

Learn

We found that we will face the same problem concerning the delay of the delivery process of the order from the airport. We also had to find alternatives to all the cell lines that were not available in our country, and beginning the transformation step.

Iteration 12:

Design and research:

After receiving some of the parts and materials needed, we faced a new problem which is the difficulty to extract and isolate mesenchymal stem cells (MSCs). This is due to the lack of antibodies and growth media essential for its characterization.

Build

We found another cell line that could be used instead of MSCs which is called Human Embryonic Kidney 293 cells(HEK 293). This cell line is considered an effective way to use viruses to evolve target genes and transfer them into the cells.

Test

Unfortunately, the HEK 293 cells could not survive in our hot temperatures even in the lab incubators. So we had to find another alternative to resume our lab work. After a quick but deep research, we finally replaced the HEK 293 with WI-38 which is a diploid human cell line composed of fibroblasts derived from lung tissue.

Learn

We finally settled on the WI-38 cell line as a good replacement for MSCs. However, we still needed to isolate the autoreactive B-cells in order to apply our design on them.

Iteration 13:

Design and research:

We found that it is impossible to isolate the autoreactive B-cells due to many reasons as:

• Ethical consideration of taking direct samples from patients.

• Lack of lab protocols needed for the isolation of only autoreactive B-cells while sparing normal B-cells.

Build

In our fourteenth iteration, we decided to make a proof of concept on anti-CD19 instead of CCP1 which is the self-antigen attacked by ACPA of the autoreactive B-cells.

Test

The anti-CD19 binded to the CD19 receptor that is overexpressed on the surface of the autoreactive B-cells. This activated the internal domain of the Syn-Notch receptor to produce the engineered exosomes. These exosomes contain the CRISPR system responsible for suppressing B-cells.

Learn

We can finally prove our concept on the CD19 receptor, as it is available and easier to implement in the lab. We now have to assure the effectiveness of DART-V-ADAR safety switch.

Iteration 14:

Design and research:

We made the DART-V-ADAR switch sensitive for ACPA mRNA, which makes it specific for autoreactive B-cells only and also incorporates the cre-recombinase system to the exosomes circuit.

Build

Due to the difficulty of isolating the autoreactive B-cells mentioned in the previous iteration, we had to make some modifications to our safety switch. Hence, instead of targeting ACPA mRNA, the switch was directed towards BAFF-R mRNA, which is found in all the B-cells, and also integrates the cre-recombinase system to control the expression of exosomes.

Test

On testing the new switch activity, we found that it is sensitive for all B-cells. This means that its specificity was decreased by our last modification. However, this was intended to happen in order to compare the sensitivity of the safety switch with other cells other than B-cells, and also we controlled the expression of exosomes in relation with binding of MSCs with the auto-reactive B-cell.

Learn

We tested the DART-V-ADAR switch sensitivity by comparing between its function on B-cells and on other types of cells, and also our new approach that control the exosome expression.

References

• Kojima, R., Bojar, D., Rizzi, G. et al. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nat Commun 9, 1305 (2018). https://doi.org/10.1038/s41467-018-03733-8.
• Sgodda M, Alfken S, Schambach A, Eggenschwiler R, Fidzinski P, Hummel M, Cantz T. Synthetic Notch-Receptor-Mediated Transmission of a Transient Signal into Permanent Information via CRISPR/Cas9-Based Genome Editing. Cells. 2020 Aug 20;9(9):1929. doi: 10.3390/cells9091929. PMID: 32825374; PMCID: PMC7563181.
• Li, M., Fan, YN., Chen, ZY. et al. Optimized nanoparticle-mediated delivery of CRISPR-Cas9 system for B cell intervention. Nano Res. 11, 6270–6282 (2018). https://doi.org/10.1007/s12274-018-2150-5
• Gayet, R.V., Ilia, K., Razavi, S. et al. Autocatalytic base editing for RNA-responsive translational control. Nat Commun 14, 1339 (2023). https://doi.org/10.1038/s41467-023-36851-z