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Background
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Project Design
Generally, the project design can be divided into three portions:
(1) RNAi module
(2) targeting module
(3) RNAi module and targeting module assembly.
RNAi module
RNA interference (RNAi) is a powerful tool that uses siRNA segments to turn off or silence the expression of specific target genes. We designed specific MOR siRNAs that can function as therapeutic agents to degrade MOR mRNA and block MOR protein expression and function through the RNAi pathway. Four siRNA sequences targeting different sites of the MOR open reading frame (ORF) were designed based on a free software accessible online. This tool can find the best siRNA sequences on the targeted gene MOR to insure the maximum gene-specificity and silencing efficacy. This tool also designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. At least two of the four pre-designed shRNA plasmids are guaranteed to knock down expression of the targeted gene MOR. The availability of two effective sequences allows proper control of non-specific and off-target effects. When the shRNA plasmids of MOR are transfected into HEK293 cells, the shRNA hairpin structure is cleaved by Dicer into siRNA of MOR. However, because siRNA is not stable in vivo and because the blood-brain barrier also prevents siRNA uptake into the neurons, carriers to facilitate siRNA uptake into the neurons need to be developed.
Figure 1. Four pre-designed siRNA sequences.
Target module
Exosomes are natural nano-sized vesicles secreted by endogenous cells. Given their intrinsic role as natural transporters of bioactive molecules between cells, exosomes potentially represent siRNA carriers for therapeutic purposes. Because exosomes can be genetically engineered and modified and are biocompatible with the immune system, they have the ability to deliver siRNA to specific cellular environments without causing cytotoxicity or the immune response. Alvarez-Erviti et al. are the pioneers of delivering siRNA drugs by this method [1]. To ensure that exosomes can load and deliver siRNAs to targeted tissues, we redesigned natural exosomes through three rounds of genetic modification, one round for outside modification and two rounds for inside modification.
Outside modification: We engineered our chassis, human embryonic kidney 293 (HEK293) cells, to express a fusion protein composed of the exosomal membrane protein Lamp2b and a neuron-targeted short peptide of RVG. Lamp2b (lysosomal-associated membrane protein 2b) is a protein ubiquitously expressed on the surface of exosomes. RVG (rabies virus glycoprotein) is a specific ligand for the acetylcholine receptor that is abundantly present on the surface of neurons. By genetically engineering the RVG peptide to the outer membrane portion of Lamp2b, Lamp2b can bring the RVG peptide to the surface of exosomes. Through these modifications, exosomes will be redesigned to specifically recognize and target neuronal cells by binding exosomal surface RVG to acetylcholine receptors on neuronal cells.
Figure 2. We connected RVG to Lamp2b using a glycine-linker to construct our fusion protein and promoted its expression by the promoter Pcmv. Then, our site-specific exosomes could deliver siRNA to brain tissue.
Inside modification I: We packaged MOR siRNA into exosomes by transfecting HEK293 cells with a plasmid expressing MOR siRNA, and then collected siRNA-encapsulated exosomes.
Inside modification II: To enhance the production efficiency of exosomal siRNA of MOR, we introduced a “molecular pump” into the cellular chassis to accelerate the amount of exosomes released by cells and to increase the amount of miRNAs packaging into exosomes. Because neutral sphingomyelinase 2 (nSMase2), a key regulatory enzyme that generates ceramide from sphingomyelin, actively induces exosome secretion from cells and triggers cellular export of small RNAs [2], we selected nSMase2 as the “molecular pump” to promote the production of exosomes and exosomal siRNAs. Thus, a plasmid designed to specifically express the full-length open reading frame (ORF) of nSMase2 was constructed and transfected into HEK293 cells to overexpress nSMase2 and to stimulate the secretion of exosomal siRNAs from HEK293 cells.
Figure 3. Function of nSMase2 molecular pump.
Assembly of the RNAi module and targeting module
By expressing neuron-targeting RVG peptide on the surface of exosomes, filling exosomes with MOR siRNA and pumping out more exosomal siRNAs from cell chassis, we will harvest a large amount of exosomes carrying MOR siRNA inside and expressing RVG peptide on their membranes. When the modified exosomes are injected into the bloodstream, the exosomes will specifically recognize acetylcholine receptors and fuse with neurons under the direction of the RVG peptide. Once inside neurons, MOR siRNA will degrade MOR mRNA by base-pairing, resulting in a sharp decrease in MORs on neuron membranes. In theory, the delivery of MOR siRNA to the targeted neurons will be achieved, whereas non-specific uptake of MOR siRNA in other tissues will be avoided. As a consequence, the reduction and disturbed function of MOR will result in the inhibition of GABA secretion and the suppression of the dopaminergic reward pathway, which ultimately will have therapeutic effects on opioid dependence.
Reference
1.Alvarez-Erviti, L. et al. Nat. Biotechnol. 29, 341–345 (2011).
2.Kosaka N, Iguchi H, Yoshioka Y, Takeshita F, Matsuki Y, Ochiya T. Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells[J]. The Journal of Biological Chemistry. 2010,285(23):17442-52.
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