Team:NJU China/Project
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Biomissile: a novel drug delivery system with exosome | Biomissile: a novel drug delivery system with exosome | ||
Recently, small interfering RNA (siRNA) has emerged as a promising therapeutic drug against a wide array of diseases. However, site-specific delivery has always been a challenge in gene therapy. Exosomes are lipid-bilayer vesicles which are naturally secreted by almost all cell types, playing crucial roles in intercellular transport of bioactive molecules. Given the intrinsic ability to naturally transport functional RNAs between cells, exosomes potentially represent a novel and exciting drug carrier. In our project we are trying to express both anti-virus siRNA within the cell and target protein on the surface of the exosomes by engineering the HEK 293T cell, which is capable of producing large amounts of exosomes. Thus, the exosomes produced by our engineered HEK 293T cells will contain the siRNA and be able to specifically deliver the siRNA to the sites we want, acting as biomissile for the targeted destruction of the disease. | Recently, small interfering RNA (siRNA) has emerged as a promising therapeutic drug against a wide array of diseases. However, site-specific delivery has always been a challenge in gene therapy. Exosomes are lipid-bilayer vesicles which are naturally secreted by almost all cell types, playing crucial roles in intercellular transport of bioactive molecules. Given the intrinsic ability to naturally transport functional RNAs between cells, exosomes potentially represent a novel and exciting drug carrier. In our project we are trying to express both anti-virus siRNA within the cell and target protein on the surface of the exosomes by engineering the HEK 293T cell, which is capable of producing large amounts of exosomes. Thus, the exosomes produced by our engineered HEK 293T cells will contain the siRNA and be able to specifically deliver the siRNA to the sites we want, acting as biomissile for the targeted destruction of the disease. |
Revision as of 17:43, 27 September 2013
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Overview
Chassis
Targeting module
Killing module
Achievement
Exosomes are lipid bilayer vesicles that 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 exosomes 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 cell 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 exosomes 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.
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Overview
abstract
Biomissile: a novel drug delivery system with exosome
Recently, small interfering RNA (siRNA) has emerged as a promising therapeutic drug against a wide array of diseases. However, site-specific delivery has always been a challenge in gene therapy. Exosomes are lipid-bilayer vesicles which are naturally secreted by almost all cell types, playing crucial roles in intercellular transport of bioactive molecules. Given the intrinsic ability to naturally transport functional RNAs between cells, exosomes potentially represent a novel and exciting drug carrier. In our project we are trying to express both anti-virus siRNA within the cell and target protein on the surface of the exosomes by engineering the HEK 293T cell, which is capable of producing large amounts of exosomes. Thus, the exosomes produced by our engineered HEK 293T cells will contain the siRNA and be able to specifically deliver the siRNA to the sites we want, acting as biomissile for the targeted destruction of the disease.
Targeting medication has always been a challenge in gene therapy. It is urgently required to develop a new system to overcome the off-target effect, low efficiency and high toxicity of the currently available approaches.
Using the principles of synthetic biology, we aimed at building up a new drug delivery system named bio-missile. We wanted to encapsulate small interfering RNA (siRNA) as a therapeutic drug into targeting exosome for site-specific delivery.
Exosomes are lipid bilayer vesicles, which are naturally secreted by almost all cell types, playing crucial roles in intercellular transport of bioactive molecules. Given their role as natural transporter, exosomes potentially represent a novel and exciting drug carrier for therapeutic purpose. Thus, modification of exosomes derived from cells may realize the goal of delivering drugs to local cellular environment.
Outside modification:
To endow the exosome with site-specific recognition ability, we designed a fusion proteins comprising of exosome surface protein lamp 2b and receptor-binding peptides. The lamp 2b can bring the receptor-binding parts of the fusion protein onto the surface of the exosome. Thus, the modified exosome will, in theory, has the ability to target specific tissues and organs.
Inside modification:
We have managed to encapsulate our ‘kill device’ siRNA into exosomes. siRNA is emerging as a promising therapeutic drug against a wide array of diseases and it functions to destroy mRNA through the RNA interference pathway. By designing siRNA against certain disease-related genes, we can use siRNA as molecular medicine for disease treatment.
By transfecting our chassis, HEK 293T cells, with siRNA plasmids and then collecting exosomes, we filled the exosomes with therapeutic siRNAs. Via the engineering of the target protein, we also endowed the exosome with the site-specific targeting ability.
Our modified exosomes are just like the ‘bio-missiles’, which can be delivered to specific cells and destroy target mRNAs, causing destruction of specific diseases. Our project will open up new avenues for therapeutic applications of exosomes as bio-missile.
Chassis
![](https://static.igem.org/mediawiki/2013/5/5d/293t_mv.jpg)
Targeting module
![](https://static.igem.org/mediawiki/2013/e/e7/Tra.png)
Killing Module
Based on the utilization of natural exosome produced by HEK 293T cells and the modification of the surface protein, lamp-2b, now we have got a site-specific drug carrier, which can bring medicine to cells with a certain kind of receptors. In order to expand power of our system, we decide to pack the carrier with our disease-killing device, siRNA.
Recently, small interfering RNA (siRNA) is emerging as a promising therapeutic drug against a wide array of diseases. siRNA functions through the RNA interference pathway. Normal double-stranded RNA is first processed by Dicer and Argo to become short double-stranded RNA, which is about 21-25bp in length. Then it will recruit other proteins to form RISC( RNA induced silencing complex). One of the two RNA stands in the RISC will be degraded and the remaining strand can specifically recognize other mRNA by base pairing. Once the RISC bind to other complementary mRNA, it will destroy the mRNA through the RNAi pathway. By designing siRNA against certain virus genes, we can use siRNA as molecular medicine for diseases treatment. siRNAs are well tolerated and have suitable pharmacokinetic properties.
![](https://static.igem.org/mediawiki/2013/2/26/RNAi_small.png)
This discovery encouraged us to harness siRNA as specific targeted drugs producing a therapeutic benefit in our system. Now, not only can we carry drugs into a specific site, but also the drug itself specifically turn off the disease-causing gene expression rather than destroy the whole cell or disrupt normal protein production in a healthy cell.
Design
We carried out this experiment in the HBV testing model to verify that our big idea can work in real human disease.
The first step in designing a siRNA for viral gene silencing is to choose the siRNA target sites.
Firstly, we should find 21 nt sequences in the target mRNA from Hepatitis b virus (HBV) genome that begin with an AA dinucleotide.
And then, choose target sites from among the ‘AA sequences’ based on guidelines like ‘Target sequence should have a GC content of around 50%’; ‘Avoid stretches of 4 or more bases such as AAAA, CCCC; ’‘Avoid regions with GC content <30% or > 60%.’
We completed the first two steps in the software, siRNA Designer.
Finally, we performed BLAST homology search to avoid off-target effects on other genes or sequences
After screening the HBV genome using the methods mentioned above, we ultimately find three candidates as our anti-HBV siRNA
siRNA 308 TATGCCTCAAGGTCGGTCGTT against HBx gene in HBV genome
siRNA 467 TCCCATAGGAATCTTGCGAAA against HBsAG gene in HBV genome
siRNA 516 ACAAATGGCACTAGTAAACTG against HBsAg gene in HBV genome
The number 308, 467 and 516 in the siRNA indicates their target sites within their target gene.
![](https://static.igem.org/mediawiki/2013/3/3c/U6_si_small.png)
We cloned these three siRNAs into the vector pENTR/U6, which is a plasmid backbone for high yield of siRNA.
By cloning the siRNAs into eukaryotic expression vectors, large amounts of corresponding siRNAs can be produced by the cell, instead of chemically synthesizing the siRNAs.
In these vectors, we use an RNA polymerase Ⅲpromoter U6 to direct the transcription of siRNA, and an enhancer which greatly increases the gene transcription
Results
1.siRNA screen
Since we have designed three types of siRNA target to different HBV genes, we need to determine which one would be optimal for HBV treatment.
To achieve this goal, we first tested their relative expression level in cells and exosomes. Expression of them was confirmed by quantitative PCR (qPCR) analysis of transfected HEK 293t cells and exosomes collected from the culture medium. The result suggested that 467 siRNA had much higher level of expression in both cells (Fig.1) and exosomes (36h post-transfection)(Fig.2). Since we need high yield of siRNA in the exosome, we decided to choose 467 as our ‘kill device’.
Fig.1 :qPCR analysis of relative siRNA level in 293t cells. Result showed that 467 siRNA has a relatively higher level of expression than that of 308 siRNA and 516 siRNA.
Fig.2 :qPCR analysis of relative siRNA level in 293t cells. 24 hours after transfection, 516 has a relatively higher expression level in exosomes. However, after 36h’s transfection, 467 performed a significant increase in expression level, almost 100 times of that of 516 and 308. Therefore, together with the data from Fig.1, we eliminated 516 siRNA and 308 siRNA from our list.
2.Silencing effect of siRNA 4667 towards HBsAG
We investigated the silencing effectiveness of siRNA 467 towards HBsAG to guarantee that it would work as we want. By qPCR analysis of HepG2 cell co-transfected with both siRNA 467 plasmid and HBsAg plasmid, we observed the significant down-regulation of HBsAg gene (Fig.3). This demonstrated that our siRNA 467 does function to silence the target gene, yet it could not prove that 467 siRNA would also work when it was encapsulated into our exosomes.
Fig. 3 :Silencing of HBsAg by siRNA 467. All groups were transfected by Lipo 2000. Control group was transfected with empty plasmid. siRNA-free group was transfected with HBsAg overexpression plasmid, and experimental group with both HBsAg overexpression plasmid and 467 siRNA plasmid. The result suggested that 467 siRNA successfully down-regulatethe HBsAg gene.
3.Silencing effect of siRNA 467 towards HBsAg after encapsulated in exosome
After transfecting 293t cells with 467 siRNA plasmids, we collected exsomes and then co-cultured them with HepG2 cells which had been transfected with HBsAg over-expression plasmid. qPCR analysis confirmed that the down-regulation of HBsAg was significant(Fig.4), and thus proved that our 467 siRNA could work effectively in exosomes.
Fig.4:
All groups were transfected by Lipo. The first group was transfected without any plasmid while other three groups were transfected with HBsAg over-expression plasmid. Second group cultured without exogenous exosomes. Third group co-cultured with empty exosomes, and fourth with exosomes containing siRNA 467. These two kinds of exosomes were collected from 293t cells transfected without and with 467 siRNA plasmid, respectively. Then every group was analyzed for their HBsAg gene expression level through qPCR. Since group 2 and group 3 showed a similar level of expression, either of which is significantly higher than group 1, the HBsAg gene did express in those cells. Yet group 4, which was co-cultured with 467 exosomes, showed a relatively lower HBsAg expression level. That may indicate that 467 contained in exosomes had down-regulated the target gene.
4.Absolute quantification of siRNA 467 encapsulated into exosome
In order to better apply our exosomes in further experiment (mice experiment, for example), we needed to absolutely quantify the siRNA productivity in each μg of exosomes (whose quantity is measured base on protein concentration). By qPCR analysis of a series of siRNA with given concentration, we drew a standard curve (Fig.5). Then using this curve, we were able to determine the siRNA quantity within exosomes. Also, we tested the quantity of siRNA from both 24h post-transfection and 48h post-transfection in each experiment. The results showed that 24 hours after the transfection, the quantity of siRNA per 1μg exosome was 8*10-2 fmol in exosomes(Fig.6). However, after 48 hours, the number nearly doubled. It was 2*10-1 fmol(siRNA)/μg(exosome) in exosomes. The results helped us determine the dose of injection for mouse and establish the mathematical model.
Fig.5 siRNA 467 Standard Curve drawn through a series siRNA with given concentration.
Fig.6 Absolute quantification of siRNA 467 in exosomes, 24h and 48h post transfection, respectively.
![](https://static.igem.org/mediawiki/2013/2/26/RNAi_small.png)
![](https://static.igem.org/mediawiki/2013/3/3c/U6_si_small.png)
![](https://static.igem.org/mediawiki/2013/c/ca/Nju_Project-4-1.png)
![](https://static.igem.org/mediawiki/2013/0/0e/Priject-4-2.png)
![](https://static.igem.org/mediawiki/2013/e/e0/NjuProject-4-3.png)
![](https://static.igem.org/mediawiki/2013/1/1d/NjuProject-4-4.png)
![](https://static.igem.org/mediawiki/2013/6/6a/M3-Project-killing-_Fig5.png)
![](https://static.igem.org/mediawiki/2013/8/8e/M3-Project-killing-_Fig6.png)
Achievement
Starting from March, our lab work continues for 7 months. During these 7 months, we first came up with the idea of ‘biomissile’ via brainstorming, then carefully designed every component of our biomissile. And the most difficult part was to experimentally prove that every part of the system can work properly. From the hard work of the past 7 months, we have successfully done the following:
We designed and created a new anti-HBV siRNA biobrick(BBa_K1180000), and experimentally validated that it can significantly suppress the HBV viral gene. Apart from that, after transfection of HEK 293T cells with this part, the siRNA can be successfully encapsulated into the exosmes produced by the cells.
We designed and constructed a liver-targeting fusion protein (BBa_K1180003) . After transfecting the HEK 293T cells with this plasmid, we monitored the exosomes with this fusion protein on its surface successfully get into the Hep G2 cell.
We designed and constructed a brain-targeting fusion protein(BBa_K1180002). After transfecting the HEK 293T cells with this plasmid, the exosomes produced by the HEK 293T cells can successfully bring the siRNA contained into the mouse brain.
Combining the targeting module and kill module together, firstly it can target specific sites, apart from that, the siRNA contained within the exosome can specifically destroy the disease related gene. Thus we realize the idea of boimissile by using our engineered exosome for target-destruction of disease.