Targeting and Delivery

Design overview

We employed the widely used exosome targeting protein RVG-Lamp2b with an engineered glycosylation site to guide exosomes to nerve cells. In addition, Cx43 (S368A) – a mutated gap juction protein, was introduced to promote the fusion and internalization of our engineered mRNA-carrying exosomes.

Aim of the design

For tefficient delivery of our drug-carrying exosomes to the brain, the modified RVG-Lamp2b is transported to the exosome membrane to enhance the passage of the exosomes facross the blood brain barrier and targeting neurons. Meanwhile, we utilized Cx43 (S368A) to help with therapeutic esmRNA release to the cytoplasm of the recipient cellsm.

Detailed introduction of the genes employed

RVG is a cell penetrating peptide (CPP) that is able to penetrate mammalian cell membranes. As a ligand of nicotinic acetylcholine receptor, RVG can recognize the blood brain barrier (BBB) and targets nerve cells. If anchored on nanoparticles, RVG can assist them in passing through the BBB by transcytosis and entering the neurons subsequently. After attaching to the nicotinic acetylcholine receptor of neurons, exosomes will be taken into cells through receptor-mediated endocytosis.

Lamp2b is one of the three variants of lysosome-associated membrane protein 2, which are membrane glycoproteins. It densely distributes on the membrane of multivesicular bodies, which can invaginate to form exosomes. We used the fusion protein of RVG and Lamp2b, which has previously been registered and demonstrated by the NJU-CHINA team, to target exosomes to the nervous system.

GNSTM is a 5-residue sequon that serves as an attachment site for N-linked glycosylation. Introduction of this sequon to a targeting peptide can add an efficient glycosylation site that can prevent the targeting peptide from degradation, without affecting the peptide-receptor interaction on the targeted cells. We fused this sequon to the N-terminus of RVG-Lamp2b for enhancement of targeting efficiency of exosomese.

Cx43, or Connexin 43, is a gap junction protein that can oligomerize to form hemichannels. As a transmembrane protein, it naturally locates on the membrane of fa majority of human cells, and also on the membrane of multi-vesicular bodies and exosomes. Cx43 is able to modulate the interaction and transfer of cellular content between exosomes and the recipient cells. Ah study (Citation) discovered that not only small molecules, but large molecules that could not pass through hemichannels, like plasmids or mRNA, can be better delivered by exosomes in the presence of highly-expressed Cx43. It was attributed to Cx43’s function of promoting membrane fusion between exosomes and recipient cells’ membrane. Based on the above arguments, we overexpressed this gene to increase the delivery efficiency of our cargo mRNA into nerve cells.

Test of the design

To test our design, two plasmids, pcDNA3.1-GNSTM-RVG-Lamp2b and pcDNA3.1-Cx43 were constructed.

Test of the construct pcDNA3.1-GNSTM-RVG-Lamp2b

Three plasmids, pcDNA3.1-GNSTM-RVG-Lamp2b, pcDNA3.1-L7Ae and pcDNA3.1-nanoluc-C/Dbox were co-transfected into HEK293T cells as the test group. In the other group, pcDNA3.1-GNSTM-RVG-Lamp2b is replaced by empty pcDNA3.1 plasmid as negative control.

Exosomes produced by two groups of the cells were collected. These exosomes were added into the culture medium of two groups of Neuro-2a cells respectively. After two hours’ incubation, Neuro-2a cells were subsequently collected, and qRT-PCR was performed to compare the concentration of nanoluciferase mRNA between two cell groups.

Test of the construct pcDNA3.1-Cx43

Four plasmids, pcDNA3.1-GNSTM-RVG-Lamp2b, pcDNA3.1-L7Ae, pcDNA3.1-nanoluc-C/Dbox and pcDNA3.1-Cx43 were co-transfected into HEK293T cells as the test group. In the other group, pcDNA3.1-Cx43 is replaced by empty pcDNA3.1 plasmid as negative control.

Exosomes produced by two groups of the cells were collected. These exosomes were added into the culture medium of two groups of Neuro-2a cells respectively. After six hours’ incubation, Neuro-2a cells were collected, and nanoluciferase assay was performed to detect two cell groups’ expression rates of nanoluciferase.

References

Alvarez-Erviti, L., Seow, Y., Yin, H., Betts, C., Lakhal, S. and Wood, M. (2011). Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature Biotechnology, 29(4), pp.341-345. doi: 10.1038/nbt.1807

Akers, J., Gonda, D., Kim, R., Carter, B. and Chen, C. (2013). Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. Journal of Neuro-Oncology, 113(1), pp.1-11. doi: 10.1007/s11060-013-1084-8

Hung, M. and Leonard, J. (2015). Stabilization of Exosome-targeting Peptides via Engineered Glycosylation. Journal of Biological Chemistry, 290(13), pp.8166-8172. doi: 10.1074/jbc.M114.621383

Soares, A., Martins-Marques, T., Ribeiro-Rodrigues, T., Ferreira, J., Catarino, S., Pinho, M., Zuzarte, M., Isabel Anjo, S., Manadas, B., P.G. Sluijter, J., Pereira, P. and Girao, H. (2015). Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells. Scientific Reports, 5(1). doi: 10.1038/srep13243