Part:BBa_K1180002

fusion protein of lamp 2b and RVG for brain targeting of the exosome

This part is designed to direct exosome to the brain. 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.For brain targeting, we choose to use RVG, which is a short peptide from Rabies Virus[1], as our target protein. RVG can specifically recognize acetylcholine receptor in the central nervous system, thus we engineer the RVG peptide into the lamp 2b .

Characterization

We got some preliminary data both in vitro and in vivo proving that RVG-exosomes can be delivered into the brain. 1.In vitro evidence for RVG-exosome entry into the neuron We used qPCR to measure the relative siRNA probe level in the neurons co-cultured with both empty RVG-exosomes and siRNA containing RVG-exosomes. We can easily see from Fig.1 that almost no siRNA was detected from the neurons co-cultured with 40 μg empty RVG-exosomes, while the siRNA detected in neurons co-cultured with siRNA probe containing RVG-exosomes show both significant and dose dependent increase.

Figure.1 The RNA was extracted from the primary cortical neurons co-cultured 24h with 40 μg empty exosome, 20μg siRNA containing RVG-exosomes,40μg siRNA containing RVG-exosomes, respectively. And the RNA extracted was measured by qPCR using probe for the siRNA encapsulated in the exosomes.

2.In vivo evidence for the entry of RVG-exosomes entry into the brain To further investigate whether the RVG-exosome can get into brain, we intravenously injected the empty RVG-exosomes, siRNA containing RVG-exosomes, respectively, into the mouse. Then we took the brain out and measure the siRNA level in the cortex and medulla. As shown in Fig.2, no siRNA was detected in both cortex and medulla after the injection of empty RVG-exosomes, while for the siRNA containing RVG-exosomes, the siRNA detected in the cortex and medulla are significant higher than that of the empty exosomes.

Figure.2 The mice were intravenously injected with 200 μg empty and siRNA containing RVG-exosomes, respectively once a day, and continued for four days. On the fifth day, the mice were killed and their brains were taken out. The RNA from their cortex and medulla were measured using siRNA probe for the siRNA encapsulated in the exosome.

References

[1]Alvarez-Erviti L, Seow Y, Yin H F, et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes[J]. Nature biotechnology, 2011, 29(4): 341-345.

Sequence and Features

Assembly Compatibility:

• 10
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  Illegal PstI site found at 832
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• 12
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  Illegal SpeI site found at 448
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• 21
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• 23
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  Illegal XbaI site found at 1135
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• 25
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  Illegal XbaI site found at 1135
  Illegal SpeI site found at 448
  Illegal PstI site found at 832
  Illegal PstI site found at 1218
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1312
  Illegal BsaI.rc site found at 150