Design

Introduction

This year, we are providing a novel therapeutic approach to meet the needs of the market and patients with autoimmune diseases generally and rheumatoid arthritis specifically and to provide personalized better management of the disease condition and overcome the adverse effects of the current methods of treatment:

SUPER (Smart Universal Pleomorphic Endogenous Regulator) of immunity

Any disease has three main treatment principles: curative, preventive, and palliative. Curative treatment of rheumatoid arthritis can be achieved in many ways according to its severity, such as lifestyle modification, medical treatments, and surgical procedures if it causes a severe joint deformity. The current treatment, which is mainly corticosteroids, causes systemic side effects such as osteoporosis, hypertension, hyperglycemia, truncal obesity, a supraclavicular pad of fat, and infections secondary to immune suppression. Even more, the second line of treatment is biological treatments such as DMARDs, which cause nausea, vomiting, abdominal pain, and hematological affection. Our aim is a complete cure for rheumatoid arthritis through “ integration between cell-based and mRNA-based therapeutic approaches” . Generally, there are considerable advantages to RNA-based therapy, such as its transient activation that does not alter the normal human body physiology. Also, it can carry the message of any gene to produce any functional protein to perform its desired specific function. mRNA-based therapy is marked by fast production in addition to being not harmful to the genetic materials. On the other hand, RNA-based therapy has low delivery efficacy, lacks specificity to its target cells, and has a limited duration of expression that would require multiple dosage therapies. Similarly, cell-based therapy has pros and cons. It has some privileges: the ability to be tuned through sensing the external environment; complexity; multi-functionality, and extended therapeutic effects corresponding to their long lifespan. However, most of the engineered cells' functions are still relatively constrained without employing their full therapeutic potential. The majority of their functions are also mediated by changing the state of the cell, even by suppression or activation, to secret specific substances such as cytokines, perforin, granzyme, etc. that spread throughout the entire body via the bloodstream, However, this usually leads to multiple undesirable systemic side effects.

SUPER is a novel approach integrating the two leading therapeutic modalities (RNA & Cell-based) through engineered mesenchymal stem cells (MSCs) that:

specifically target the auto-reactive B-cell and express modified exosomes containing our cargo in the form of mRNA coding for the CRISPR-Cas system designed to knock down the B-cell activating factor receptor (BAFF-R) gene that is mainly expressed in B cells to maintain their activity and survival.

The SYN-notch receptor that regulates the expression of the mRNA cargo is designed to sense the external environment of the (MSCs). In our condition, this receptor is designed to specifically sense the presence of autoreactive B-cells secreting the anti-citrullinated peptide antibodies (ACPA), which is the most specific marker for rheumatoid arthritis . Moreover, these antibodies titer reflects the severity and progression of the disease. Therefore, we have chosen auto-reactive B-cells that secrete ACPAs to be our biomarker to tune the activity of SUPER to cope with the disease remission and progression phases. This means the more auto-reactive B-cells, the more mesenchymal stem cells will be stimulated through the SYN-notch receptors. Unlikely, coping with disease progress is difficult to manage with the current methods of treatment. Concisely, this privilege ensures that our SUPER is an adjustable platform that acts according to the disease condition and severity of each patient.

The integration of the previous two modalities has allowed us to overcome the limitations , such as low efficacy and specificity and unwanted systemic side effects, by using MSC to deliver our therapeutic agent-CRISPR-Cas system, targeting BAFF-R in the form of mRNA loaded in exosomes. These exosomes are secreted and assembled within our engineered MSCs to target the autoreactive B-cells that secrete ACPA to stop its pathogenic effect within the body. As the MSCs are viable, they will keep producing exosomes that will lead to a longer half-life time of the therapeutic agent to cope with disease progression in the body in comparison to using purified exosomes that are characterized by short half-life and will need multiple doses for better effect.

We enhanced the specificity of our approach based on MSCs through:

• Adding a SYN-notch receptor specific for auto-reactive B-cells allows us to link the expression of the therapeutic agent to the levels of the auto-reactive B-cells and correspondingly boosts the expression of the therapeutic agent loaded in the exosomes.

• Adding CCP1 antigen as the extracellular receptor of the exosome will increase the possibility of delivering our cargo to the target cells, due to the high affinity between the CCP1 and the recognition site of the autoreactive b-cell.

• Adding a tissue-specific switch called DART-V-ADAR (Detection and Amplification of RNA Triggers via Adenosine Deaminase Acting on Double-stranded RNA) before the coding sequence allows the transcription of the downstream sequences. That happens only in the presence of ACPA mRNA of the auto-reactive B-cell, which will limit the off-targeting events and ensure that our therapeutic agent carries out its effect in the proper target cells.

This will make our “ SUPER” platform personalized for every patient, as it will be tuned by the activity of the SYN notch receptor, which is mediated by the number of autoreactive B-cells in the environment. As a result, there will be less chance of complications, such as multiple organ damage, in the later stages of the disease due to improper management of the patient's condition. In addition to controlling the cycles of relapse and progression that are poorly managed by the current treatment modalities.

Elements of the design

1-Synthetic notch:

Syn notch is a modular receptor based on the core regulatory domain of mechanosensitive notch receptors (mouse notch) combined with an adjustable artificial sensor (extracellular ligand binding domain) and transcription mediator within the internal domain.

In our design, this receptor consists of three main elements:

• An extracellular ligand binding domain represented in the cyclic citrullinated peptide that has a high affinity to the variable region of ACPAs.This domain transmits the tension force generated from the binding with the auto-reactive B-cell receptor.

• A transmembrane domain mouse notch core protein which is the mechanosensitive portion of the Syn notch receptor as the mechanical force delivered from the external domain carries out a confirmatory structural change that induces proteolytic cleavage in the S1 portion of the negative regulatory region (NRR) in order to transmit the binding signal to the next component.

• An Intracellular domain that contains our transcription activation module VP64 combined with zinc finger peptide 21.16. This ZF21.16 directs the activity of VP64 toward the ZF21.16 minCMV promoter that controls the transcription of our therapeutic agent. This combined part (ZF21.16-VP64) is released after the proteolytic cleavage of the transmembrane domain to induce the expression of our therapeutic agent.

The presentation of our syn-notch receptor on the surface of MSCs is mediated by CD8 alpha signal.

--For more details visit the Parts page.

2-Exosomal element

We have chosen the exosomes secreted from MSCs to be our biological endogenous vector for our therapeutic agent, so we had to implement some modifications to increase their effectiveness in delivering our cargo selectively to the auto-reactive B-cells secreting ACPA with minimal off-targeting effects.

This is performed by adding:

A) Exosomal receptor:

To enhance the affinity of the exosomes toward the autoreactive B-cells, we expressed CCP1 antigen that has a high binding affinity to the recognition site of the auto-reactive B-cell receptor on the exosomal surface. The presentation of CCP1 on the exosomal membrane is done by combining with Lysosome-Associated Membrane Glycoprotein 2b (LAMP2b) which is a transmembrane protein specific to exosomes and lysosome membranes, so the possibility of exosomal B-cell fusion is markedly increased.

B) Booster genes
This part is responsible for enhancing the default levels of exosomes secretion to magnify the numbers of exosomes containing our therapeutic agent and improving the effectiveness of SUPER to eliminate the auto-reactive B-cells. These booster genes are represented in (STEAP3, SDC4, and NadB) . They regulate three certain pathways: exosome biogenesis, the budding of endosomal membranes to form multivesicular bodies and cellular metabolism. This overall increases the production of exosomes.

We improved this composite part by controlling the expression of booster genes through integrating the Cre loxP system. This system consists of two elements: first, loxP-(STOP)-loxP located upstream to the coding sequence of booster genes. Therefore, it hinders the translation of our booster genes.The second Cre recombinase enzyme is expressed conditionally following the activation of CCP1-syn-notch receptor.

This enzyme will trigger the transcription of our booster genes by deleting the upstream STOP sequence. As a result, the expression of booster genes is now conditioned by the presence of Cre recombinase enzyme.

For more details, you can visit the Parts page.

C) Loading system
Loading our therapeutic agent in the form of single-stranded RNA is mediated through a loading system consisting of two domains:

• RNA binding protein (L7Ae) conjugated to the intramembrane domain of CD63, which is a whole-mark antigenic protein naturally highly expressed on the surface of exosomes.

  

• C\D boxes is the RNA hairpin structures that are located in the 3` end of our cargo that bind to L7Ae to ensure loading of our therapeutic cargo within the exosomes.

  

D) Connexin
Connexin (CX43) is a transmembrane protein that mediates intracellular communication. We implicated this part to enhance the entrance of our cargo into the exosomes to be loaded within it.

For more details, you can visit Parts page

3-Cargo:

A) CRISPR-Cas system targeting BAFF-R gene

CRISPR-Cas system was discovered as a part of the adaptive immunity in most bacteria and has been manipulated in different ways to be one of the most effective and leading systems for gene editing in synthetic biology.

In our case, we used Cas12k protein, as it is considered one of the shortest members of the CRISPR family system that can effectively fit in the exosomes, which have low loading capacity. Cas 12k editing activity is directed through complementary single-stranded RNA with a certain protospacer adjacent motif (Pam) sequence. Cas12k recognizes mainly DNA, but it also has the ability to detect RNA by incorporating reverse transcription. In our design, the guide RNA is designed to drive the activity of Cas12K toward BAFF-R in the autoreactive B-cells to impair its survival and activity. As the interaction between crRNA and the target DNA activates the endonuclease within the RuvC domain that cleaves the BAFF-R gene.

For more details visit the Parts page.

B) DART V ADAR switch

DART-V-ADAR is a novel programmable tissue-specific sensor established on base editing technology and its activity depends on the adenosine deaminase enzyme acting on double-stranded RNA (ADAR) that has the capability to convert mismatched adenosine (A) groups into inosine (I) through a hybridization process.

The sensor of this switch is a modular single-stranded RNA designed to be complementary to RNA specific to the target tissue, which is the mRNA of ACPA that presents in the autoreactive B-cells. This sensor is designed to maintain the switch in the off state as it contains a stop codon (UAG) preventing the translation of the whole strand unless it's deaminated by ADAR activity if the sensor finds its complementary strand. Then, the adenosine (A) base in the stop codon will be converted to inosine (I), changing the state of the switch to the on state.

To amplify the signal transmitted from the initial reaction of the switch in the on state, we introduced exogenous copies of ADAR enzyme within the cell through conditional expression of a mutant form of “ADAR2” gene and conjugation of the catalytic domain of it to the RNA binding domain of MS2 coat protein (MCP), which will drive the activity of MCP-ADAR to the sensor sequence flanked by two MS2 hairpin structures. Therefore, this system acts as a positive feedback loop to amplify the signal after delivery of the cargo to the cell of interest.

Integrating this approach into our design allows us to gain high specificity and increase safety by minimizing the off-target effects that could be mediated by the exosomal delivery system.

For more details, you can visit Parts page.

4-suicide gene

We integrated the IC9 system as a suicide gene to manage MSCs' possible adverse effects, such as fibrosis and thromboembolism, by inducing apoptosis of the cell after administration of CID (chemical inducer of dimerization) that induces apoptosis of the transferred MSCs by the IC9 system.

For more details visit the Safety page

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