Introduction

Biosafety and security is considered one of the most important aspects in synthetic biology. It is concerned with the safety of all working team members by providing them a safe environment with minimal lab hazards, and injuries. Therefore, we were trained on general lab safety before starting our wet lab phase. Furthermore, the safety of the consumer is one of our major priorities that could be achieved by applying molecular safety techniques to prevent the potential hazards of engineered cells. So, we designed our project with several safety layers to prevent expected hazards from our SUPER-MSC.

Why MSCs?

We are introducing a new cell-based therapy for rheumatoid arthritis. This treatment depends on engineered mesenchymal stem cells (MSCs) to deliver our CRISPR-Cas system for the target auto-reactive B cell loaded on exosomes secreted by engineered MSCs. MSCs are the best choice for us to deliver our CRISPR system, because of an innate immune-modulatory function as MSCs have a great role in the regulation of immunity during various inflammatory responses including auto-immune diseases. In addition to that, MSCs have a group of chemokine receptors on their surface that are responsible for their migration and adhesion to the site of inflammation to ensure that MSCs only do their immune-modulatory function in the presence of inflammation. This adhesion is facilitated by molecules for adhesions like Integrins, Cadherins, Selectins, and CD44 that also have a great role in cell signaling. When the MSCs arrive at the site of inflammation where there are a lot of Extracellular Matrix (ECM) components that interact with the receptors on the MSCs. These interactions facilitate MSC migration and adhesion. Also, they regulate its function by regulating the diffusion of some growth factors by this they can change its response to environmental cues. According to that, the MSCs is our best choice to use in our project because of their innate immune-modulatory function and natural safety package mentioned with it.

Possible hazards:

Addressing the project’s safety approach in relation to different parts used in the genetic circuit:

First problem

After a lot of research, we found some literature mentioning that cell-based therapy using MSC increases the chance of developing tumors or worsens the case of already presented ones, especially connective tissue tumors.

First solution

So, in order to solve this possible risk, we had to implicit the use of a suicidal gene: IC9 system into our engineered MSCs. As, it was previously proven to be effective and has been already used in similar projects that utilize stem cell-based therapy. IC9 is a suicide system that will give us the ability to terminate the life of our MSC selectively through apoptosis. The apoptotic death and suicide of the MSC will occur in case of the liability of uncontrolled proliferation and the possibility of the conversion of the MSC into cancerous cells. The IC9 system is composed of 2 main inactive-domains that are activated upon exposure to a chemical inducer of dimerization (CID). CID is an external non-toxic element that activates the IC9 system and induces our MSC apoptosis terminating its effect on the patient’s immune system.

Second problem

Using exosomes as a vehicle for delivery is very promising, as exosomes have a high capability to be engineered and modified by multiple approaches in addition to their ability for fusion and endocytosis with the cells. This is due to the presence of fusogens expressed on the exosomes’ surface. But this can also represent a weak point in our case, as our design is based on delivering CRISPR -Cas system targeting B cell activating receptor (BAFF-R) genes within the autoreactive B cells only, This BAFF-R gene is essential for the B cells' survival.

So the high capacity of exosomes to deliver our system will be met by a lack of specificity and selectivity. Accordingly, we had to integrate and take multiple measures to limit the off-targeting capability and effect that could be mediated by the use of a non-selective exosomal delivery system.

Second solution

Overview

Our solution is mainly based on the expression of a Cas12k system, in response to the binding of Syn notch receptor to the BCR-external domain (B-cell Receptor), representing the ACPA (Anti-citrullinated peptide antibodies) of the autoreactive B-cells. Upon which the internal domain of the Syn notch receptor will induce the release of Cas12k mRNA that will then be loaded on an exosomal-based delivery system. These exosomes loaded with Cas12k mRNA will recognize the autoreactive B-cells by engineered exosomal receptors. Following the binding between the engineered exosomal receptors and the BCR of the B-cells, endocytosis of the exosomes into the B-cells will occur. Then the CRISPR-Cas12K-mRNA cargo will be released inside the autoreactive B-cells. This Cas12K-mRNA is already engineered by adding a synthetic safety tissue-specific switch, called (DART-V-ADAR). This switch is an RNA sequence that perfectly matches the RNA sequence (Anti-Citrullinated Peptide Antibody) ACPA-mRNA inside the B-cells, except for one base pair that is intentionally placed prior to the Cas12k-mRNA sequence, in the switch that we call DART-V-ADAR. This mismatching Adenosine base pair is inserted into a STOP codon (UAG), that will trigger the ADAR enzyme. This enzyme is responsible for the replacement of the mismatching adenine base pair that is part of a stop codon in the DART-V-ADAR switch preceding the Cas12g-mRNA sequence. Therefore the Adenosine base pair will be replaced by another nucleotide sequence matching the pairing between the DART-V-ADAR sensor and the ACPA-mRNA, removing the Stop codon thus stopping the inhibitory action of the DART-V-ADAR switch on the expression of the Cas12k. Accordingly, the Cas12k mRNA will be translated and act freely on the BAFF-R gene, causing apoptosis of the autoreactive B-cells.

Safety Measures: Step one (Syn-notch & BCR interactions):

We designed a modular therapeutic system: This system conditionally releases an exosomal-based delivery cargo, on the condition of stable binding between syn-notch receptor & BCR (autoreactive B-cell receptor). Therefore the release of the loaded exosomes depends on the presence of auto-reactive B-cells. So, it will be a personalized therapy for each patient according to the severity of the disease. Reducing the off-targeting risk of our therapeutic system: As our system won’t be activated unless the autoreactive B cell is found within the surrounding media.

Safety Measures: Step Two (Cargo-loaded Exosomal delivery system):

Ensuring the specific delivery of the cargo-loaded exosomes to the targeted auto-reactive B-cells:

To avoid the

false recognition

Safety Measures: Step Three Tissue-specific switch (DART-V-ADAR):

Delivering our therapeutic agent in the form of mRNA and hindering it’s translation through adding a novel tissue-specific switch to the cargo thus Cas9/g/BAFF-R won’t be translated until it is present within the targeted B cell, the idea of this system is based on ADAR enzyme capability in base editing for double-stranded RNA so the switch designed to contain a sensor with a stop codon in its sequence and complementary to specific RNA present in the targeted cell or tissue, therefore, we engineered the sensor to be complementary to part of ACPA mRNA which is specific for the autoreactive B cell. So if our system successfully transferred to the autoreactive B cells the sensor will be in the on state and the stop codon (UAG) will be altered to (UIG) by ADAR enzyme activity and our Cas9/g/BAFF-R will be translated to carry out its function.

Delivering our therapeutic agent (cargo) loaded within the exosomes that consists of 3 main delivered elements:

• Tissue-specific safety switch: Detection & Amplification of RNA-triggered via ADAR enzyme (DART-V-ADAR)

• CRISPR Cleaving system: Cas12k/g mRNA

• Signal magnifier: catalytic portion of ADAR2 enzyme conjugated to MS2 coat protein(MCP-ADAR2).

Cas12k/g mRNA is preceded by DART-V-ADAR switch that inhibits the translation except in the auto-reactive B cell by the following mechanism: The DART-V-ADAR contains a single-stranded RNA sensor that is complementary to the variable domain of the ACPA mRNA, except in a single adenosine group within a stop codon (UAG) pair that will be mismatched with its opposite base pair. This stop codon inhibits the translation until the sensor binds to its complementary RNA strand (ACPA mRNA) within the target auto-reactive B-cells. So if our system successfully transferred to the autoreactive B-Cell and the sensor binds to its complementary RNA the ADAR enzyme changes the mismatched Adenosine in (UAG) into Inosine (UIG). Therefore the stop codon is disturbed and the translation of the Cas12k/g mRNA can be resumed.

This step in our design is considered the most effective approach in our safety measures to tackle the problem of exosomal off-targeting effect as it is based on the innate function of the ADAR enzyme and its ability to edit RNA base pairs. Therefore, it needs minimal modification to be used in other applications just by changing the sequence of the sensor to be complementary to the specific mRNA that is present only within the targeted cell or tissue.

There are three different ways to design this system:

• The sensor could be designed with no exogenous source of ADAR.

• Constitutive overexpression of exogenous supplementation of ADAR.

• Conditional expression of ADAR.

We have chosen the third option by adding ADAR2 enzyme to the circuit of our cargo that is expressed conditionally following the activation of the Syn notch switch, thus the frequency of editing events will be markedly higher. This ensures a higher dynamic range and improves the switch performance in other copies after the initial reaction takes place within the target cell by forming a positive feedback loop and amplifying the signal from endogenous ADAR.

To enhance the sensitivity and efficiency of ADAR2 enzyme we added the catalytic portion of ADAR2 enzyme within the circuit of our cargo conjugated to MS2 coat protein (MCP) to improve the functionality of the DART-V-ADAR switch. MCP-ADAR2 is acting as a signal magnifier for our therapeutic agent through creating a positive feedback loop that improves the chance of translation of other copies of our cargo transferred into the target auto-reactive B-Cell.

Tutorial for DART-V-ADAR sensor design

First step:

You have to determine specific mRNA within the target cell or tissue according to your project to be the target of the sensor.

second step:

Choose a CCA site in the DNA sequence of the target RNA and select 48 base pairs on both sides to make a total of 99 base pairs.(note: Best CCA sites are in 3'UTR.)

Third step:

Write the reverse complement of the previous 99 base pairs.

Fourth step:

Edit the central TGG reverse (complement of) CCA into TAG that codes for stop codon UAG.

Fifth step:

Select two blocks of 7 base pairs located 24 bp upstream and 23 bp downstream of TAG.

Sixth step:

Replace the previous blocks with MS2 hairpin sequence.

Seventh step:

Insert the reverse complement in the sensor cloning site flanked by two P2A linkers in your circuit according to your design, but ensure that the sensor precedes the coding sequence of interest that needs to be regulated.

Notes:

• Ensure that the reading frame in the sensor is free of other stop codons than the central TAG.
• Edit any start codon within the sensor.
• The final length of the sensor is 123 bp long.

Third problem

We found that the overproduction of exosomes through constitutive expression of booster genes (SDC4, STEAP3, NadB) may contribute to the development of numerous side effects as allergic reactions to modified exosomes and their atherosclerosis development. as it was found that exosomes inhibit the activity of the cholesterol epoxide hydrolase which cleaves the cholesterol 5,6.

Solution

To prevent the possible hazards that may occur due to boosting the exosomal secretion, we improved a composite part designed by (iGEM18_LZU-CHINA) by implicating Cre-loxP system for gene editing. Cre-loxP system is formed of two main elements that allow us to conditionally express our booster genes following the activation of our syn notch receptor targeting the auto-reactive B-cells: The first element is loxP-STOP-loxP sequence located upstream of our booster genes to block their expression. These loxP sites orientation and sequence direct the second element activity. The second element is the Cre recombinase enzyme which is expressed under the control of ZF21.16 minCMV promoter in case of our receptor activation. This enzyme performs its recombination activity within the sequence flanked by two loxP sites. In our case, Cre recombinase deletes the STOP sequence that blocks the translation. This deletion occurs because loxP sites are placed in the same direction upstream and downstream of the STOP sequence.

General safety principles

PDF for General safety principles

References

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