Overview

This year, we are addressing scarless wound healing. This depends on increasing skin regenerative abilities, which can be achieved through cells and proteins. We decided to exploit MSCs’ regenerative feature, meanwhile, adding a novel protein that is involved in regulating cellular growth, proliferation,and differentiation. This protein is called yes-associated protein (YAP-1). Our project is confined to 3 systems:

1. A synthetic receptor system which is composed of an external domain receptors (VEGFR-1 and VEGFR-2) targeted to a tissue injury biomarker called : Vascular endothelial growth factor (VEGF), transmembrane domain, and internal domain which is divided into two separate chains. Each chain carries a tobacco etch virus protease (TEV) , its cleavage site (TCS), and dcas-9 domain.
2. A loading system which carries the YAP-1 mRNA inside the exosomes, delivering mRNA to other cells. This system consists of CD-63 and MCP.
3. A YAP-1 specific mRNA switch that stimulates YAP-1 mRNA translation only in presence of an intracellular tissue injury biomarker called matrix metalloproteinase-9 (MMP-9). This system consists of MMP-9 nanobodies, YAP-1, HHR, NSP3, and MS2-aptamers.

Fig 1. Action vedio bftth fg [7].

Receptor

Our engineered MSCs possess a receptor that is specifically designed to recognize VEGF , a protein whose levels rise significantly in response to tissue damage. When this receptor binds to VEGF, it triggers a signaling pathway that leads to an increase in the intracellular levels of YAP-1 within the MSCs. This elevated YAP-1 level motivates the MSCs to proliferate and differentiate into various cell types found in the local wound environment, thereby promoting tissue regeneration.

Our receptor is formed of two separate longitudinal chains. Each chain consists of components from the three domains of the receptor( external , transmembrane, and internal). Thus, we divided TEV protease and dCas9 into two separate components, each in a separate chain.

1. First Chain
• External Domain

Part Name	Description	Type
VEGFR-1	It is a vascular endothelial growth factor receptor which is classified into 3 types of similar structures. Their external parts are made up entirely of repeating segments that resemble parts of antibodies (immunoglobulin homology repeats) and they have a critical role in the formation of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis).	Basic

• Internal Domain

Part Name	Description	Type
C-TEV	This is known as the tobacco etch virus which is very selective for cleaving proteins at particular amino acid sequences.Moreover, it has been modified to have better qualities like greater heat stability, and lesser autolysis (self-cleavage). Additionally, it allows researchers to precisely cleave the tag off, leaving behind the receptor in its unmodified form. In our model, TEV was divided into N-terminal and C-terminal fragments. So, the C-terminal fragment was grafted onto dCAS9(C) to generate TEV(C)-dCas9(C).	Basic
NLS	A nuclear localization signal which is a short sequence of amino acids. NLS is found in certain proteins in which it acts as a zip code, directing those proteins to the nucleus of a cell. In our model, it is attached to dCas9(C) to keep it in the nucleus, preventing dcas9 self assembly.	Basic
TCS	This is the TEV cleavage site, a particular amino acid sequence detected by TEV to start cleavage.	Basic
d-Cas9 (C)	A dead Cas9 is a modified version of the CRISPR-Cas9 gene editing tool. Unlike CRISPR-Cas9, dcas-9 can’t cut DNA. Instead, it can bind to a specific DNA sequence, guided by an RNA molecule,to activate or suppress the target gene. In our model, dcas9 was divided into N-terminal and C-terminal fragments. So, the C-terminal fragment was grafted onto NLS to generate NLS-dCas9(C).	Basic
VP-64	It is a strong transcriptional activator composed of four tandem copies of VP16 (Herpes Simplex Viral Protein 16) connected with glycine-serine (GS) linkers. When fused to another protein domain, it binds near the gene promoter.	Basic

2. Second Chain
• External Domain

Part Name	Description	Type
VEGFR-2	It is a vascular endothelial growth factor receptor which is classified into 3 types of similar structures. Their external parts are made up entirely of repeating segments that resemble parts of antibodies (immunoglobulin homology repeats) and they have a critical role in the formation of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis).	Basic

• Internal Domain

Part Name	Description	Type
N-TEV	This is known as the tobacco etch virus which is very selective for cleaving proteins at particular amino acid sequences.Moreover, it has been modified to have better qualities like greater heat stability, and lesser autolysis (self-cleavage). Additionally, it allows researchers to precisely cleave the tag off, leaving behind the receptor in its unmodified form. In our model, TEV was divided into N-terminal and C-terminal fragments. So, the N-terminal fragment was grafted onto dCAS9(N) to generate TEV(N)-dCas9(N).	Basic
NES	Nuclear Export Signal is a short sequence of amino acids found in certain proteins that direct proteins outside the cell nucleus. In our model, it is attached to dCas9(N) to keep it outside the nucleus, avoiding dCas9 self assembly.	Basic
TCS	This is the TEV cleavage site, a particular amino acid sequence detected by TEV to start cleavage.	Basic
d-Cas9 (N)	A dead Cas9 is a modified version of the CRISPR-Cas9 gene editing tool. Unlike CRISPR-Cas9, dcas-9 can’t cut DNA. Instead, it can bind to a specific DNA sequence, guided by an RNA molecule,to activate or suppress the target gene. In our model, dcas9 was divided into N-terminal and C-terminal fragments. So, the N-terminal fragment was grafted onto NES to generate NES-dCas9(N).	Basic

To sum up, our receptor is confined to two separate chains. When an injury occurs, extracellular VEGF levels increase and bind to our receptor causing the dimerization of TEV protease. This leads to the cleavage of the receptor chains at the TCS site, resulting in dimerization and release of dCas9. The dCas9, complexed with VP64, is then guided by our guide RNA to target the Nanog gene, promoting VP-64 transcriptional activation and leading to increased YAP-1 expression, respectively.

Part Name	Description	Type
Nanog gRNA1	Guide RNA is a crucial component of the CRISPR-Cas9 gene editing system as it acts as a dCas9 guide to its target. It consists of two main parts. Firstly, Spacer Sequence: which is a short, user-defined sequence that complements (matches) the target DNA sequence. Secondly, Scaffold Sequence which is pre-designed RNA structure allows gRNA to bind to the Cas9 protein and facilitate its interaction with the target DNA.	Basic
Nanog gRNA2	Guide RNA is a crucial component of the CRISPR-Cas9 gene editing system as it acts as a dCas9 guide to its target. It consists of two main parts. Firstly, Spacer Sequence: which is a short, user-defined sequence that complements (matches) the target DNA sequence. Secondly, Scaffold Sequence which is pre-designed RNA structure allows gRNA to bind to the Cas9 protein and facilitate its interaction with the target DNA.	Basic
Nanog gRNA3	Guide RNA is a crucial component of the CRISPR-Cas9 gene editing system as it acts as a dCas9 guide to its target. It consists of two main parts. Firstly, Spacer Sequence: which is a short, user-defined sequence that complements (matches) the target DNA sequence. Secondly, Scaffold Sequence which is pre-designed RNA structure allows gRNA to bind to the Cas9 protein and facilitate its interaction with the target DNA.	Basic

Receptor Composites

Part Name	Description	Type
First chain of Receptor (VEGF-R1,C-TEV, NLS, TCS,d-CAS9(C), HA, VP64)	In our first receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R1, is attached to a protein sequence containing TEV protease, a nuclear localization signal (NES), a TEV cleavage site(TCS), and dCas9(C). When an injury occurs, VEGF levels rise outside the cell. This binds to VEGF-R1, activating the receptor and triggering the TEV protease to cleave the chain at the TCS site. This releases dCas9(C), which can then be associated with a complementary dCas9 fragment (dCas9(N)) to form a fully functional dCas9 enzyme.	Composite
Second chain of Receptor (VEGF-R2, N-TEV, NES, TCS, d-CAS9(N))	In our second receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R2, is attached to a protein sequence containing TEV protease, a nuclear export signal (NES), a TEV cleavage site(TCS), and dCas9(N). When an injury occurs, VEGF levels rise outside the cell. This binds to VEGF-R2, activating the receptor and triggering the TEV protease to cleave the chain at the TCS site. This releases dCas9(N), which can then be associated with a complementary dCas9 fragment (dCas9(C)) to form a fully functional dCas9 enzyme.	Composite

Our Switch

To mitigate the potential adverse effects associated with YAP-1 overproduction, we developed a switch that controls its translation. This switch comprises several key components: a cap with NSP3 bound to an MMP-9 Nanobody; a coding region encoding the YAP-1 protein; an MS2 element linked to MCP and another MMP-9 Nanobody; and an HHR element attached to the poly A tail.

Fig 1. gauze none bftth fg [7].

Fig 2.

References

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	Description	Value	Units	Reference
K1	Rate of expression of external domain (CCP1) Syn-Notch receptor	0.001	day-1 × cell-1	[1]
K2	Reciprocal rate of degradation of MSCs	15	Days-1	[2]
K3	Rate of formation on Syn-Notch receptor on MSCs	0.033	Days-1	[1]
K4	Rate of the binding state between S and B	0.000411	day-1 × cell-1	[3]
K5	Reciprocal rate of degradation of autoreactive B-cell	30	Days	[4]
K8	Rate of dissociation of the binding state	0.001	day-1	[-]