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Team:NJU China/Project
From 2013.igem.org
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Overview
Chassis
Exosomes are lipid bilayer vesicles which are secreted by all cell types, and its diameter ranges from 30nm to 100nm. Given its role as a natural transporter of bioactive molecules, we want to utilize exosome as our drug carrier. The first problem we met is which chassis to choose to produce the exosomes we want. After screening through a large amount of different cell types, we choose to use HEK 293T cells as our chassis. HEK 293T cells is a subtype of human embryonic kidney cells and we choose this as our chassis for three main reasons. The first reason is that HEK 293T cells can secrete large amounts of exosomes so we can get enough exosomes by using HEK 293T cells as our chassis. The second reason is that HEK 293T cells are derived from human, so the exosome they secrete will be more human compatible and have little chance of inducing immune response compared to other non-human cells. The last reason is that HEK 293T cells are immortalized cells, which means that after genetically engineering them to produce the exosomes we want, we can simply subculture the cell line and keep them as cell factory to produce our desired exosomes massively.
Targeting module
Specificity is one of the most vital goal which many different drug delivery system want to achieve, and that is also exactly what we are expecting from our system. To achieve specificity, we need to make our exosomes able to recognize distinctive sites within the body. In order to do that, the first step we took towards our exosome is outside modification. We want to add a target protein onto the surface of the exosome to endow it with specifity, therefore we need to first find a protein naturally expressed on the surface of the exosomes and then fuse our target protein to the membrane protein of exosomes. After a round of screen of the different membrane proteins on the exosome’s surface, we choose to use lamp 2b to construct our fusion protein. Lamp 2b (lysosomal associated membrane protein 2b) is a protein ubiquitously expressed on the surface of the exosomes. By genetically engineer a target protein to the outmembrane part of the lamp 2b, we can use the lamp 2b to bring our target protein to the surface of the exosome. Thus we can endow the exosome with site-specificity by the addition of the fusion protein. As the target protein in the lamp 2b is an exchangeable part, we can use different target protein to direct our exosomes to different parts of human body. Liver，T cells and especially brain are poor targeting sites for previous drug delivery system. In order to prove that our target module does work, we choose to target these sites for testing. Liver: For liver targeting, we need to first find a protein specifically recognize hepatic cells. Since Heptitis B virus can infect hepatic cells distinctively, and from recent study[], we know that HBV recognizes the hepatic cells via the interaction between the pre-S1 of the HBV envelop protein and NTCP receptor of the hepatic cells. We tried to engineer the pre-S1 from HBV envelope protein to the lamp 2b. Therefore we cloned the pre-S1 into lamp 2b, and we choose pcDNA 3.1(+) as our backbone.
Killing device
To be added.
Application
To be added.
