Cells and gene therapies are two pioneering technologies in the medical field. They hold tremendous potential for a great transformation in the medical field through their modular and dynamic capabilities, offer great promise for treating a range of untreatable diseases, and personalize patients’ treatment, unlike traditional conventional pharmaceutics (1).
Moreover, regenerative medicine and tissue engineering were transformed from the phase of biomaterials development to the field of multiple approaches integration as scaffolds, cells, and biologically active substances. They aim to restore and improve damaged tissues or even whole injured organs, which is the optimum goal of medicine (2).
However, the recent therapeutic modalities based on regenerative medicine still lack sensitivity and specificity; moreover, adaptation to different environments still needs further studies to control stem cells’(SCs) destiny - which cell line they would differentiate into (3).
Therefore, to improve the regenerative capabilities of SCs, we have implemented synthetic biology. Moreover, we have linked their activity levels to the injury severity through specific tissue injury biomarkers, ensuring the dynamicity of the SCs' performance (4).
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This year, we have developed a new modular platform known as SCs-based Occlusive Nutritive Gel of Healing (SONG-H) which combines multiple technologies including: SCs, mRNA-based therapeutics, and tissue engineering. For local delivery of our SCs we have used a hydrogel scaffold as a method for loading, we have chosen skin burn injuries as a prototype to prove our project’s concept.
Furthermore, we took the stability, sensitivity, and specificity of our platform into concern as we designed a novel modular receptor relying on a new version of CRISPR dCas9 technology for signal transduction that displays minimal OFF-state baseline activity and robust ON-state ligand-induced signal transduction based on intracellular transcription mediator release using tobacco etch virus(TEV) protease module, besides that we developed a novel customized device that precisely controls and regulates the activity of trans genes, significantly improving the safety and effectiveness of gene-based therapy.
Treatment options for burns are classified according to the size and severity of the burn. Regarding first and superficial second-degree burns, conservative treatments and painkillers like ibuprofen are usually recommended. On the other hand, deep second and third-degree burns, that reach full thickness, require invasive reconstructive surgeries, as the wound cannot heal on its own. The most common surgeries for such burns are skin grafts and flaps. In recent years, new treatment protocols have emerged, such as film-based dressing, hydrogel-based wound dressings, bio-engineered skin grafts, bio-printing-based strategies, nanotherapeutics, and stem cell therapy. These treatments have shown promising results, nevertheless, further research and development are still required (1,2).
Despite the significant advancements in burn treatment and management, there are still many limitations in the available therapeutic options. For instance, deep and large surface area burns can be life-threatening and challenging due to the massive tissue damage and loss that increase the wound's susceptibility to be affected by multiple complications such as infection, scarring, contracture, and chronic neuropathic pain(3).
This year, we are targeting one of the most challenging aspects of burn injuries: disfiguring scars. It has been recently managed through invasive surgical approaches such as skin grafting and skin flaps which have multiple limitations such as the relative incompatibility between large surface area burns and lack of skin donation sites besides the risk of graft rejection. Moreover, injured area closure by grafting carries a high risk of developing contracture. As a consequence of the previous drawbacks and restraints, we decided to fill the gap in the current therapeutic approaches and develop a new promising method to overcome these barriers that hinder burn patients from acquiring effective, safe, non-invasive, and personalized non-dose-dependent management that ensure better physical and psychological outcomes for patients.
Grade 2B and 3 burn injuries are characterized by fundamental damage to cells, tissues, and vascular destruction.
Therefore, the inflammatory reaction following burn injury is relatively massive compared to non-burn trauma. Hence, the inflammatory mediators secreted from immune cells such as (TGF-B1, TNF-A, IL-6, IL1-B, and VEGF) can exaggerate the localized inflammatory response into a systemic one. Furthermore, structural proteins and growth factors were detected to be highly dominant locally and even systemically such as MMPs(1,2,3,8,9), HGF, IGFBP-1, and VEGF (4,5).
We can use multiple sensitive biomarkers to reflect the extent of tissue damage and the severity of the wound. This allows us to equip our platform (SONG-H) within MSCs to control their activity by sensing the external environment and sending a signal to the cell to perform the required function in accordance with the pre-designed system targeting the genes of interest.
In recent years, a new interest in regenerative medicine and stem cell-based renewal finds the possibility of integrating tissue engineering and gene therapy into the field, which aims to synthesize genetically modified cells that can inhibit proteins’ over-production, and repair cell functions that may optimize their therapeutic potentials for multiple diseases. In addition to that, cell-based gene therapy has shown superiority over viral-based approaches for transfecting genetic materials to the target tissue or cells due to its safety, effectiveness, and dynamicity in treating a wide variety of genetic and acquired diseases.
Therefore, significant efforts are being invested in non-viral vectors to enhance their efficiency by implementing new technologies: plasmid-based expression system that encodes a therapeutic protein and synthetic gene delivery devices.
This year, we AFCM iGEM 2024 team has focused our efforts on developing a novel modular dynamic system to enhance the performance of cell-based therapeutics and tissue engineering and to push the safety limits of cell-based gene therapy to new boundaries. We have taken skin burn injuries as a proof of concept for our innovative platform stem cell-based occlusive nutritive gel of healing (SONG-H).
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[3] Fang T, Lineaweaver WC, Sailes FC, Kisner C, Zhang F. Clinical application of cultured epithelial autografts on acellular dermal matrices in the treatment of extended burn injuries. Ann. Plast. Surg. 2014;73:509–515. doi: 10.1097/SAP.0b013e3182840883
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We have met our goals by implementing two powerful tools into SCs. These tools are capable of sensing different target molecules within the cells’ environment and performing diverse programmed responses.This is why they can achieve adaptability by stringent off/on state transition and can offer ligand-mediated dose-dependent therapeutic response.
Mesenchymal stem cells have attracted great attention as an influential method for tissue regeneration. They’re considered an ideal cell source for regenerative medicine due to its excellent properties like their ability to be extracted from different tissues including bone marrow, and adipose tissue. Besides, their capability to differentiate into multiple cell lines as osteoblasts, chondrocytes, dermal fibroblasts, keratinocytes, and endothelial cells. Likewise, their immunological properties include anti-inflammatory, immunoregulatory, and immunosuppressive capacities, contributing to their potential role as immune-tolerant agents (1,2).
Therefore, MSCs are considered the best candidates for our project since they would offer a massive attribution to our design which optimizes the wounds’ healing process.
MSCs perform their proliferative and immunoregulatory effect by intercellular communication through various ways including cell-to-cell contact, soluble molecules, and exosomes that mediate different cellular responses according to their target and the state of the surrounding environment. In addition to the role of MSCs, we can improve their role and enhance the condition of the wound by the combination of using stem cells with hydrogels as a wound dressing (2).
In addition to MSCs' previous features including direct and indirect effects, their exosomes carry a great potential to resemble an intercellular messenger due to their ability to transport different mediators such as DNA, RNA, miRNA, and proteins.
They are extracellular spherical lipid bilayer vesicles ranging from 40-150 nm in diameter.
The release of the exosome’s content into the recipient cell cytoplasm performs alterations in intracellular signaling pathways to modulate cellular processes and functions.(3)
Despite the great potential of MSCs and their exosomes in regenerative medicine, they still lack specificity and sensitivity to respond in proportion to the injury condition and severity.
As a result, major consequences and adverse effects may occur corresponding to long-term use-associated risks such as tumor formation, fibrosis, and thromboembolism. Moreover, regarding the local application of MSCs and their exosomes on injured skin, it carries relative risk of developing hyperkeratosis and psoriasis-like manifestation due to their upregulated uncontrolled activity.(4,5)
As a way to prevent this misregulation, we decided to analyze the burn injury microenvironment to select sensitive biomarkers reflecting the condition of the injury in order to link the magnitude of the tissue injury to the regenerative activity of MSCs.
Hydrogels are polymers containing up to 96% water; they ensure adequate wound hydration, Considering their ease of application and disassembly, and patient comfort, the design and modification of hydrogel dressings make them valuable and beneficial for various applications, as Hydrogel dressings may be alternative to cooling burn wounds, and in large extent of burns Hydrogel may function as a temporary support, facilitating cellular processes such as adhesion, proliferation, and differentiation for the formation of new tissue. Now there are many types of Hydrogels and they can be classified based on different factors. Classification of hydrogels depends on the materials (polymers) involved, the source of the polymers, the crosslinking method, their response to stimuli, and their ionic charge. Polymers involved in the hydrogels are natural, synthetic, or a combination of natural and synthetic polymers(6).
According to sources, it is classified into ( Natural or Synthetic Polymers or combinations). Synthetic polymer hydrogels have lower biological activity than natural hydrogels, Also Synthetic polymers are not environmentally friendly because the produced solid waste material could not be degrading biologically, so we preferred natural polymer hydrogel in our project.
Hyaluronic acid (HA) hydrogel is primarily comprised of endogenous HA which is a natural polymer that is widely distributed in the human body and plays a significant role in numerous physiological processes such as cell migration, tissue hydration, scaffolds, antibacterial activity, promotes re-epithelization, collagen-fiber arrangement, and wound healing. HA hydrogel can enhance drug stability, enabling controlled drug release, and facilitating targeted delivery, It is demonstrated that the HA hydrogel loaded with stem cells provided an optimized 3D microenvironment, enhancing the therapeutic efficiency of stem cell-based therapies, and other Studies have shown that a hyaluronic acid hydrogel systems increase wound angiogenesis and normal tissue remodeling, and significantly increase the expression of growth factors and cytokines that promote female FVB/NJ mice with deep second-degree burns healing(7).
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Our approach (SONG-H) is developed for treating burn injuries through two main interacting systems :
Synthetic receptors
Engineering cells with synthetic receptors are becoming a key driver of synthetic biology innovation and have made significant progress in multiple fields as the cells become customized to sense different molecules and different inputs and respond with various outputs. Such as therapeutic molecules in the case of treating diseases and reporter genes in the condition of whole-cell biosensors(1,2,3).
In the early years, the core of the programmable synthetic receptors idea was based mainly on facilitating the communication between the cells and their external environment through linking and rewiring the native internal pathways or artificial signal transduction modalities with native or artificial signal sensing domains.(4)
So in the case of the orthogonal receptor when both the signal sensing domain and signal transducing domain are artificial such as (SynNotch) the normal cross-talk between cells won’t influence the performance in input-to-output transduction in comparison to the other receptor exhibiting normal signal sensing domain that can be easily affected by the cross-talk between cells. Therefore, the modality of synthetic receptors, depending on the native external signal sensing domain, would be an ideal candidate for us to tune the regenerative performance of our engineered MSCs, thus MSCs would be able to sense the magnitude of the injury through detecting the native biomarkers which rise after tissue damage as an intercellular communication process.(3,5,6)
dCas9 Syn-RTK
Consequently, we have engineered a new system for signal sensing and transduction called dCas9(N/C) Syn-RTK
Receptor tyrosine kinases (RTKs) are a well-characterized class of single transmembrane-domain receptors that play essential roles in regulating a variety of cellular functions. Most members of this family share a conserved receptor topology, respond to extracellular growth factor signaling, and are activated by ligand-induced dimerization. Therefore, we reasoned that it's an ideal choice which enables a vast spectrum of choices and applications through rewiring the normal cellular information flow (7).
Continuously, we have chosen dCas9 as our signal transduction model depending on its ability to regulate the transcription activity of multiple genes. dCas9 can either induce or suppress the target gene expression level according to the attached transcription factor activity (8).
The release of dCas9 from the internal domain is mediated by tobacco etch virus protease (TEV) activity
The initial design of the receptor (TMt-NLS-dCas9-TF) was done by (Toni A. Baeumler.2018). It was reported to have robust activation of target gene expression both in the presence and absence of TEV protease showing unexpected OFF-state basal activity, this basal activity was related to the breakdown and reassembly of the nuclear membrane in the rapidly dividing cells that allows ectopic activity of the wild type (WT) Cas9 on the target gene, that was reported in previous studies.
Developing of dCas9(N/C) Syn-RTK
Avoiding the drawbacks of the dCas9 Syn-RTK initial design, we have adapted multiple changes that would ensure stability and specificity of the receptor activity to reduce the OFF-state background noise.(9)
First, a new technique based on splitting dCas9 into N-terminal fragments and C-terminal fragments has been implemented and changed the design of the receptor into different two chains of the receptor TMt-NES-dCas9(N)
This design depends on the spontaneous self-assembly of the dCas9(N/C) fragments
In addition to that, the dCas9(N)
Furthermore, we adopted a new technique that regulates the activity of the (TEV) protease through linking its activity to the receptor condition by also segregation the enzyme into to N- and C-terminal inactive fragments and reassembled by ligand-dependent receptor dimerization converting them into a catalytically active enzyme in consequence The splitting of TEV protease was done through grafting of the N-TEV and C-TEV fragments upstream of NES-dCas9(N) and NLS-dCas9(C)-TF, respectively, via a flexible linker to be integrated and contained within the receptor structure. This modification would guarantee a self-contained tightly controlled signal release mechanism from our dCas9(N/C) Syn-RTK.(9)
We adopted a new technique that regulates the activity of the (TEV) protease through linking its activity to the receptor condition by also segregation the enzyme into to N- and C-terminal inactive fragments and reassembled by ligand-dependent receptor dimerization converting them into a catalytically active enzyme in consequence The splitting of TEV protease was done through grafting of the N-TEV
These previous modifications were done to enhance the ON/OFF state transition of the receptor and the evaluation of this system has shown minimal OFF-state activity and limited TEV-independent target gene induction even in rapidly dividing cells such as HEK293K.(9)
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dCas9(N/C)-TF Syn-RTK device is characterized by the ability to be easily customized by simply changing the signal sensing domain to sense the required biomarker and redesigning the dCas9 associated gRNA to direct the activity of the system toward the required site according to the gene of interest which perform the desired function. furthermore, different transcription regulating factors could be combined with the dCas9 platform allowing the induction or suppression of the target genes to certain limits based on the variable potentials of the transcription factors.(1)
The components of our receptor :
Regarding the microenvironment of burn injuries, the degree of tissue damage and inflammation makes it very rich in multiple biomarkers in different concentrations and levels that fluctuate along the process of wound healing till wound closure . However, the selected biomarker should reflect the condition and stage of the wound in addition to the damage severity. (2)
Moreover, the biomarker should be available in adequate concentration showing significant changes in its levels along the different phases of the healing process to be detected by our version of dCas9 Syn-RTK system to regulate MSCs' regenerative functions.
We have chosen VEGF to resemble our biomarker due to its distinguished characteristics including the early onset of expression following the injury in comparison to the other molecules. Moreover, VEGF concentration is considered more than enough to stimulate the activity of our system along the healing process.
Experimental studies reported the essential role of VEGF as an inflammatory mediator whose concentration was linked to the degree of tissue injury, have supported our choice of VEGF as the biomarker of interest. Therefore, we rewired the native signaling pathway of the VEGFR within our MSCs through the implementation of the dCas9 Syn-VEGFR system in order to link the severity of the injury to the magnitude of (YAP-1) expression.
We have implemented VEGFR1/2 heterodimers as the extracellular signal sensing domain constructing two different chains. This choice is based on the experimental comparison between different constructs of a synthetic receptor expressing VEGFR1
Despite that, there was still a concerning hypothesis regarding the possibility of dimerization of the native VEGFR and our synthetic chains that would lead to basal activity and leakiness within the system.
This hypothesis was evaluated and adjusted by performing multiple techniques that maximize system performance by tuning the kinetic activity of the TEV protease. Hence, multiple mutated variants of the TEV Cleavage Sites (TCS)
Grafting The mutated variants of TCS altered the sensitivity and the kinetic activity of the TEV protease significantly that would allow us to tune the receptor to be activated in a sensitive dose manner according to the lowest customized concentration of the biomarker that could activate the receptor. That suggests that dCas9-SynRTK could respond to different biological conditions even in pathological and normal states.(4,5)
Regarding our design, we are implementing a mutant deactivated form of CRISPR/Cas9(dCas9) which is considered the leading tool for gene regulation either by suppression or induction according to the coupled transcription factor (TF) and effector proteins that have been fused to dCas9 structure such as (VP64
Clustered regularly interspaced short palindromic repeats (CRISPR) was originally identified in the immune system of bacteria, with the function of destroying the invading macrophage DNA by enzymatic digestion. This system has been developed into a highly efficient gene editing and regulating tool.(6,7)
Despite the increasing adoption of these platforms due to their programmability the difference between their potentiality to induce gene expression is still under research.
These platforms are still incompletely characterized as most studies using dCas9-based TF have focused on a single type of effector domains and this can make the selection of the optimal transcription factors needed to achieve a specific experimental outcome technically difficult. Therefore we intended to test three different modalities for transcription activation of endogenous genes.
dCas9 vp64
GAL4
Later studies showed CMV enhancer
Different constructs of transcription activation models were designed using the three previous modalities in different ways to analyze the various gene induction potentials for each one of them, allowing us to choose the best option for achieving effective, safe, and maintained induction of YAP-1 protein gene to reach therapeutic non-harmful levels within the cells in a rapid onset depending on the rate of transcription activation device to induce the expression of the target gene.
1-CRISPR/dCas9vp64
2-CRISPR/dCas9(vp64+GAL4)
3-CRISPR/dCas9(vp64+GAL4)-UAS trans CMV enhancer
The performance of the previous constructs was measured through mathematical modeling simulating the kinetics of the different transcription activation candidates based on implementing experimental parameters reported by (Irwin Davidson.2019) as they were comparing the potential of the previous transcription activators to induce the expression of 10 different endogenous genes.
Our models have allowed us to analyze each candidate onset to reach their highest potential for transcription activation and the actual transcription capabilities for each construct. Furthermore, models allowed us to study the sustainability of the different transcription activators and their consistent effects.
CRISPR/dCas9(vp64+GAL4)-UAS trans-CMV enhancer model showed the highest and most sustainable levels of transcription activation and the second highest onset to reach the peak of their effect following CRISPR/dCas9vp64 model. Thus CRISPR/dCas9(vp64+GAL4)-UAS trans-CMV enhancer would be the best candidate for our platform SONG-H to induce safe effective expression of endogenous YAP-1 reaching up to thirty-fold over the normal level(11).
Moreover, we considered their overshooting side effects that may elevate the concentration of YAP-1 to reach toxic levels that may interfere with our project's safety measures. Hypertrophic effects of YAP-1 were detected when its concentration reached more than fiftyfold over normal conditions as reported by Peter Julius(12).
CRISPR/dCas9(vp64+GAL4)-UAS trans-CMV enhancer module works through the integration between three different transcription activators.
Vp64 and GAL4 complex were fused to © fragment of dCas9 sequence and trans-CMV enhancer will be attached to © dCas9 VP64/GAL4 through the addition of the upstream activation sequence (UAS) of GAL4 that will mediate the assembly of the triplet transcription activator model.(11)
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In order to direct the activity of CRISPR/Cas-based system gRNAs are required as its design is based on identification of the gene of interest.
gRNA classically is formed of two components including the 5` end constant part which is gRNA scaffold that mediates the fusion of gRNA to Cas9 protein and second part is gRNA spacer resembling the customized portion of the gRNA according to the target gene sequence.
According to our design, the gRNA is engineered to direct the dCas9(N/C) Syn VEGFR-1\2 toward two separate targets, one is an endogenous gene while the other is a synthetic transfected circuit.(1)
Therefore we have adopted a new CRISPR technology expressing multiple guide RNA encoded by one transcript.
Expressing mixed identity gRNA targeting different sites simultaneously is called CRISPR multiplexing that would offer us the ability to exploit the full potential of our dCas9(N/C) Syn-RTK system. One of the gRNA multiplexing strategies is the co-expression of multiple gRNA encoded in a single transcript separated by hammerhead ribozyme (HHR)
Regarding our project aiming to enhance the stem cells’ regenerative capabilities, we have chosen Yes Associated Protein 1 (YAP-1) gene
(YAP), also known as YAP-1 or YAP-65, acts as a transcriptional coactivator with PDZ‐binding motif (TAZ) and is recognized as the key mediator controlling the Hippo signaling pathway that regulates the transcription of downstream target genes modulating cell differentiation and proliferation condition.(2)
The yes-associated protein 1 (YAP-1) and the transcriptional coactivator with PDZ-binding motif (TAZ) are transcriptional coactivators, members of the Hippo signaling pathway, that were initially discovered in Drosophila in 1995. YAP-1 has two major isoforms YAP-1, which contains one WW domain, and YAP-2, which contains two WW domains.(3,4)TAZ, a 43 kDa protein, is different from YAP-2 in that it lacks the SH3‐BM proline‐rich region found in the 65 kDa YAP-1 protein and only has one WW domain presenting. The common structural elements found in YAP-1/TAZ include the WW domain, C‐terminal transcriptional activation domain, and TEA domain‐containing sequence‐specific transcription factors (TEAD) binding region at the N‐terminus. In terms of mechanism, activated YAP-1/TAZ translocates from the cytoplasm to the nucleus, where it binds to TEAD in the chromosome, thereby regulating the expression of target genes and exerting diverse effects. Thus, YAP-1/TAZ is closely associated with enzyme activity, cell proliferation, differentiation, tissue, and organ growth and can mediate the production of pro-inflammatory factors. All have a transcription activation domain at the C‐terminal, including PDZ binding and Coiled‐coil region. Compared with the TAZ, the YAP-1 also has a SH3 binding region and YAP-2 has one more WW domain.(5)
In fact, there are many methods for YAP-1/TAZ regulation such as Hippo signaling, wnt signaling, G protein-coupled receptor signaling, estrogen signaling and the most common regulation method is post-translation modification. Moreover, post-translation modification including phosphorylation, acetylation, methylation(6), and phosphorylation and non-phosphorylation of YAP-1/TAZ are the most common forms of their function. Phosphorylated YAP-1/TAZ is limited to the cytoplasm and degraded, while phosphorylated YAP-1/TAZ can undergo nuclear translocation and bind to TEAD in the nucleus. The phosphorylation of YAP-1/TAZ can be divided into Hippo‐dependent ways and Hippo nondependent ways. For the Hippo‐dependent way, the activated LATS1/2 directly targets YAP-1/TAZ to phosphorylate and inactivate it(7). For the Hippo nondependent ways, YAP-1/TAZ can serve as a target of multiple molecules, such as AMP‐activated Protein Kinase (AMPK), β‐AR, and mTORC2. Under the action of these molecules, YAP-1/TAZ is inhibited or activated, which affects inflammation, cancer, metabolism, organ fibrosis, and organ regeneration.
The second target is the transfected circuit expressing a modified version of YAP-1 cloned into the Translation Initiation Device(TID). The final construct of TID will be loaded in RNA form within the MSCs’ exosomes. These exosomes will transfect YAP-1 to the nearby cells within the microenvironment of the wound.
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[5]Webb C, Upadhyay A, Giuntini F, et al. Structural features and ligand binding properties of tandem WW domains from YAP and TAZ, nuclear effectors of the Hippo pathway. Biochemistry. 2011;50(16):3300–3309.
[6]Yu Y, Su X, Qin Q, et al. Yes‐associated protein and transcriptional coactivator with PDZ‐binding motif as new targets in cardiovascular diseases. Pharmacol Res. 2020;159:105009.
[7]Ma S, Tang T, Probst G, et al. Transcriptional repression of estrogen receptor alpha by YAP reveals the Hippo pathway as therapeutic target for ER(+) breast cancer. Nat Commun. 2022;13(1):1061.
Synthetic biology contains a variety of regulatory switches that allow gene expression modulation at transcriptional, post-transcriptional, translational, and post-translational levels. Recently translation-based gene regulation systems have attracted tremendous interest. As they offer us multiple features including fast sense and response dynamics besides the ability to sense a wider variety of intra-cellular signals(1).in contrast, transcription-based gene regulation systems show limited options for signals and the lag between the sense and response process(2). Therefore, we have adapted a new well characterized device acting as a translation regulation platform which is known as Translation Initiation Device (TID) providing safe conditioned expression of any mRNA transcripts.
(TID) was implemented in SONG-H to enhance its performance as a cell-based gene therapy by offering precise regulation of the transgene activity which is critical to optimize the efficacy and safety of our approach.(3)
TID is based on reprogramming one of the native mechanisms known as the closed loop model that regulates the translation of mRNA into protein within the eukaryotic cells(4,5).
The closed loop model was supported by numerous studies reporting that the transformation of mRNA into circular form is favorable for mRNA scanning and translation. This circular form is mediated through co-operative binding between poly-A tail binding protein (PABP) and cap-binding proteins including (elF4E, eIF4A, and eIF4G) approximating the poly-A tail
TID regulates the translation of reprogramed transcript through simulating the mechanisms of the closed loop model by genetically encoded replacement of the natural poly(A) signal with synthetic RNA binding protein (RBP)-specific aptamers providing proper control over translation initiation process as it will depend strictly on the presence of specific native or synthetic biomarkers(7). in consequence, the circularization of mRNA would be mediated through cooperative binding of the aptameric region within the 3` end and the eIF4F complex at the 5`-cap simultaneously to the biomarker of interest.(8)
The final construct of this system is considered as an intracellular customized protein sensing switch that offers great features providing a great opportunity to clinically apply gene based therapies reinforced by safety, sensitivity, and programmability to meet the biomedical requirements of any biological drug.
TID was manipulated to fit into our approach to enhance our engineered MSCs' performance as a platform for cell-based gene therapy.
TID is integrated with an Roading system to construct a modular self contained device for cell-based gene therapy via exosomal delivery.
1- The first component is the gene encoding for YAP-1 and its 3` end fused to the aptameric component of TID replacing the poly-A tail signal. We have chosen MS2 aptamer due to the high binding stability to their RBP MS2 coat protein (MCP).(9).
2- The second part encodes for MCP-MMP9/(NB1) that replaces PAMP.
3- The third part encodes for MMP9 (NB2) /NSP3A.replacing the cap binding protein.
[1]Ausländer, S., Ausländer, D., Müller, M., Wieland, M. & Fussenegger, M. Programmable single-cell mammalian biocomputers. Nature 487, 123–127 (2012).
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[7]Saito, H. et al. Synthetic translational regulation by an L7Ae–kink-turn RNP switch. Nat. Chem. Biol. 6, 71–78 (2010).
[8]Quintás-Cardama, A., Kantarjian, H. & Cortes, J. Flying under the radar: the new wave of BCR–ABL inhibitors. Nat. Rev. Drug Discov. 6, 834–848 (2007).
[9]Cao, J. et al. Light-inducible activation of target mRNA translation in mammalian cells. Chem. Commun. 49, 8338–8340 (2013).
The second and third parts of TID contain specific sensors (nanobodies) sensitive to MMP9 which is a structural protein that shows significant intracellular increase following tissue injury such as burns thus the transition of the TID from OFF to ON state will be conditioned by the presence of MMP9 within the cell cytoplasm mediating circularization of YAP-1 mRNA through simultaneous cooperative binding of the second and third component of the system to the target biomarker MMP9 providing favorable condition for mRNA scanning and translation of the effector protein (YAP-1) (1).
Transfected YAP-1 will promote the multiplication of the native cells of the wound besides enhancing the regenerative capabilities of the basal cell layer within the wound microenvironment.(4)
The delivery of the effector gene YAP-1 will be done by delivering YAP-1 mRNA to the cells of interest within the wound through endogenous loading and assembly of the cargo into the genetically modified exosomes equipped with an RNA loading system that is based on RBP and its aptameric sequence interaction.
TID's initial design was assessed through measuring the expression level of the grafted gene within TID.TID performance evaluation was performed in the absence and presence of the initiator biomarker(5). In consequence, the result analysis reported high off-state basal activity that would affect the regulatory mechanism of our device.
Continuously we assumed that our device leakness would be due to the presence of the poly-A tail attached to the effector transgene’s RNA at the 3`.
Poly-A tail preserving could mediate spontaneous circulation of our effector gene’s transcript to achieve the normal closed-loop model favorable for mRNA scanning and translation. In other words, the closed-loop model could be achieved even with the absence of the initiator biomarker, thus the regulatory function of TID would be significantly impaired(2).
Therefore, we had to implement a new strategy targeting the excision of the poly-A tail while keeping in mind its crucial role in mRNA stability.
Provided that, we make use of an RNA motif known as Hammer Head Ribozyme (HHR). This motif catalyzes RNA self-cleavage reactions that would be ideal for the poly-A tail excision from the 3` end of the effector gene’s transcript. Hence, we grafted the (HHR) motif just upstream to the poly-A signal (3) to separate the poly-A tail from our effector transgene’s RNA following its translation.
To optimize the performance of TID we had to tune its sensitivity toward our initiator biomarker (MMP-9). Hence, we intended to design our platform to exert a safe, effective, and dynamic therapeutic response according to the degree of tissue injury. We implemented a new technique altering the response of the TID to be concentration-sensitive. This technique is based on tailoring the binding affinity of MCP
Continuously we built 4 different constructs of TID with different MS2(×N) and evaluated their activation score in response to contact concentration of the initiator biomarker.Inconsequence, we concluded that there is a direct proportion relation between MS2 (×N)
We have generated multiple nobodies targeting MMP9 by abstracting the complementary determining region (CDR) from native antibodies of MMP9 and we tested their binding stability with MMP9 by using prodigy haddock online software tool. Nanobodies(NB) are the smallest possible form of antibodies. (NB) attracted our interest due to their high modularity in various biotechnological applications.
Recently (NB) can be generated from naïve or synthetic antibodies libraries by grafting the complementarity-determining regions (CDR) within the conventional region of the antibody format onto nanobody frameworks.
Nanobodies’ generation go through two steps:
- Collection of Framework
The VHH framework is derived from the variable domains of camelid heavy-chain-only antibodies. This framework act as the structural backbone for the nanobody. Furthermore, it offers stability and facilitates antigen binding.
- Grafting of CDRs onto the VHH Framework
CDRs are extracted from traditional antibody formats then attached to the VHH backbone. As a result, weak antigen binders are produced, which can then be utilized as templates. These templates are used to create libraries that enhances binding specificity for the target antigen.
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[2]Shao, J., Li, S., Qiu, X. et al. Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells. Cell Res 34, 31–46 (2024).
[3]Ausländer, S. et al. A general design strategy for protein-responsive riboswitches in mammalian cells. Nat. Methods 11, 1154–1160 (2014).
[4]Dey A, Varelas X, Guan KL. Targeting the Hippo pathway in cancer, fibrosis, wound healing and regenerative medicine. Nat Rev Drug Discov. 2020 Jul;19(7):480-494. doi: 10.1038/s41573-020-0070-z. Epub 2020 Jun 17. PMID: 32555376; PMCID: PMC7880238.
[5]Quintás-Cardama, A., Kantarjian, H. & Cortes, J. Flying under the radar: the new wave of BCR–ABL inhibitors. Nat. Rev. Drug Discov. 6, 834–848 (2007).
We implemented multiple modifications on MSCs’ exosomes to enhance their capabilities as an intercellular messenger to transport our effector gene (YAP-1) in the form of mRNA to the nearby cells within the microenvironment of the wound.
Firstly we equipped the exosome membrane with an RBP known as MCP that has a high affinity to its specific stem-loop RNA aptamer (MS2).
MCP was added to exosome membrane mediated by combining its sequence to CD63 is endosome-specific tetraspanin that is highly concentrated in ILVs and hence enriched in exosomes.
The loading of YAP-1’s mRNA is mediated through the fusion of MS2 aptamers to the 3` end of YAP-1 that will guarantee selective loading of YAP-1’s mRNA within the exosomes expressing MCP-CD63 complex
The final design of our therapeutic platform for burn injury consists mainly of two interacting systems that will be incorporated into MSCs through the transfection of four different plasmids coding for all system components.
Our approach works through topical application of our HA hydrogel scaffold loaded with the genetically engineered MSCs.continously, their regenerative function will be promoted through VEGF-dependent activation of the synthetic receptor “dCas9(C/N)-TF Syn-VEGFR-2/1” thus TEV proteolytic activity will mediate the release of (N) and (C) fragments of dCas9 to spontaneously assemble into one effector complex.The activity of the effector complex will be directed toward two separate targets by two different gRNAs. The first gRNA directs dCas9 to YAP-1 as it's designed to be compatible with Nanog enhancer located upstream to the YAP-1 coding sequence. Moreover, the second gRNA will direct dCas9 toward our transgene coding for a modified version of YAP-1 engrafted into TID that regulates YAP-1 expression beside its loading within the modified exosomes. These exosomes will be secreted from MSCs to transport our cargo to the neighboring cells.
In consequence, YAP-1 will increase in both our engineered MSCs and the native cells within the wound. Subsequently, YAP-1 effect would enhance the differentiation, proliferation, and migration of both MSCs and the native cells within the wound. Hence the injured tissue healing process will be significantly optimized leading to effective wound closure and restoration of the skin integrity. In other words our platform offers a new programmable technology relying on cell based therapy and tissue engineering to enhance the normal healing process in non-regenerative organs, especially skin. Furthermore, our new platform could be abstracted by other teams targeting multiple fields to serve their purpose through manipulating our parts library.
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