Team:NJU China/Project/Brain

From 2013.igem.org

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<h1>Liver: <span>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[1], we knew 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.
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<h1>Brain： <span>For brain targeting, we chose to use RVG, which is a short peptide from Rabies Virus, as our target protein. RVG can specifically recognize acetylcholine receptor in the central nervous system[2], thus we engineer the RVG peptide into the lamp 2b and we use pcDNA 3.1(+) as our vector.</span></h1>
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Therefore we cloned the pre-S1 into lamp 2b, and we choose pcDNA 3.1(+) as our backbone.</span></h1>
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<p>Site-specific drug delivery 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. Recently, small interfering RNA (siRNA) is emerging as a promising therapeutic drug against a wide array of diseases. Current techniques for siRNA transfer use viruses or synthetic agents as delivery vehicles. However, these approaches are toxic and low-efficient, and more importantly, can not deliver siRNA to specific tissues and organs. </p>
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<p>Brain targeting Results：</BR>
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To produce the exosomes that have RVG on their surface for brain targeting, we first transfected the exosome-producing cells, HEK 293T cells, with the plasmid encoding the fusion protein of lamp 2b and RVG peptide.</BR>
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To check if the RVG-containing exosomes can target brain, we use a siRNA as probe. We first encapsulated the siRNA into the RVG-modified exosomes by direct transfection of the HEK 293T cells with siRNA probe.</p>
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<p>To better deliver the siRNA and other drugs, we are trying to construct a novel system which employs MVs to encapsulate and protect siRNAs for delivery to target cells. Microvesicles (MVs) are lipid bilayer vesicles which are naturally secreted by almost all cell types, playing crucial roles in intercellular transport of bioactive molecules, including siRNAs. Given the intrinsic ability to traverse biological barriers and to naturally transport functional siRNAs between cells, MVs potentially represent a novel and exciting drug carrier for therapeutic uses. Thus, MVs derived from cells engineered to express siRNAs may be capable of delivering siRNAs to local cellular environment.</p>
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<p>1.Quantification of siRNA contained in the exosomes</BR>
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    We first quantify the amount of siRNA encapsulated into the exosomes. We transfected the HEK 293T cells (transfected with RVG plasmids before) with siRNA before collecting the exosome. We used the exosomes collected from the HEK 293T cells (transfected with RVG plasmids before) without transfection of siRNA as negative control.</BR>
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    By quantitative PCR analysis of a series of siRNA with known concentration, we drew a standard curve. By referring to this curve, we calculate the absolute amount of siRNA in the exosomes. As shown in Fig.1, the amount of siRNA in the negative control is quite low (background) while the siRNA contained in the RVG exosomes transfected with siRNA probe reaches as high as 0.8 fmol (RNA)/μg (exosome).</p>
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<p>To achieve site-specific siRNA delivery, we will express targeting protein onto the surface of MVs and the modified MVs will, in theory, have the ability to target specific tissues and organs. By transfecting the producer cells with siRNA plasmids and collect MVs, we will fill the MVs with therapeutic siRNAs.</p>
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<img src="https://static.igem.org/mediawiki/2013/8/83/NJU_M3M_BRAIN.png">
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<p>2. In vitro evidence for the entry of pre-S1 exosomes into the hep G2 cell</BR>
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As shown in Fig.3, by labeling the exosomes with DiI-C16 (red) and hep G2 nucleus with DAPI(blue), it can be seen that the exosomes successfully get into the hep G2 cells</p>
<p>Our modified MVs are just like the “biomissile”, which can be delivered to specific cells and destroy target mRNAs. Our project will open up avenues for therapeutic applications of MVs as biomissile.</p>
<p>Our modified MVs are just like the “biomissile”, which can be delivered to specific cells and destroy target mRNAs. Our project will open up avenues for therapeutic applications of MVs as biomissile.</p>

Revision as of 12:55, 26 September 2013

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Brain： For brain targeting, we chose to use RVG, which is a short peptide from Rabies Virus, as our target protein. RVG can specifically recognize acetylcholine receptor in the central nervous system[2], thus we engineer the RVG peptide into the lamp 2b and we use pcDNA 3.1(+) as our vector.

Brain targeting Results：
To produce the exosomes that have RVG on their surface for brain targeting, we first transfected the exosome-producing cells, HEK 293T cells, with the plasmid encoding the fusion protein of lamp 2b and RVG peptide.
To check if the RVG-containing exosomes can target brain, we use a siRNA as probe. We first encapsulated the siRNA into the RVG-modified exosomes by direct transfection of the HEK 293T cells with siRNA probe.

1.Quantification of siRNA contained in the exosomes
We first quantify the amount of siRNA encapsulated into the exosomes. We transfected the HEK 293T cells (transfected with RVG plasmids before) with siRNA before collecting the exosome. We used the exosomes collected from the HEK 293T cells (transfected with RVG plasmids before) without transfection of siRNA as negative control.
By quantitative PCR analysis of a series of siRNA with known concentration, we drew a standard curve. By referring to this curve, we calculate the absolute amount of siRNA in the exosomes. As shown in Fig.1, the amount of siRNA in the negative control is quite low (background) while the siRNA contained in the RVG exosomes transfected with siRNA probe reaches as high as 0.8 fmol (RNA)/μg (exosome).

2. In vitro evidence for the entry of pre-S1 exosomes into the hep G2 cell
As shown in Fig.3, by labeling the exosomes with DiI-C16 (red) and hep G2 nucleus with DAPI(blue), it can be seen that the exosomes successfully get into the hep G2 cells

Our modified MVs are just like the “biomissile”, which can be delivered to specific cells and destroy target mRNAs. Our project will open up avenues for therapeutic applications of MVs as biomissile.

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