Overview
Our project consists of two therapeutic systems (targeting system, siRNA release system), two control systems (Tet-On system and Cre-loxP system) and an exosome booster system. Taken together, the systems will operate through layer-by-layer assembly, which will form a three-layer strategy for conditional release and targeted delivery of anti-cancer exosomal siRNAs in vivo.
The first layer is the Tet-On system which is the switch of the latter two layers. This system consists of an albumin promoter and the Tet-On system. In the presence of doxycycline, the first layer will be switched on in hepatocytes and open the second layer. The second layer consists of the Cre-loxP system, the targeting system and the siRNA release system. Three siRNAs and exosome-targeting peptide iRGD can be encoded here in order to achieve the goal of targeted therapy. Meanwhile, the Cre-loxP system can turn on the expression of the third layer. .The third layer consists of exosome release system, which promote the liver to produce more exosomes to improve the efficiency of our siRNA delivery.
Systems
2.1 siRNA release System
KRAS mutation is found in approximately 40% of of patients with NSCLC. Yet no KRAS inhibitors has gone beyond clinical testing so far, making the gene deemed as “undruggable” for a long time. [1] Here we challenge the KRAS mutation lung cancer with a strategy of triple targets: the KRAS gene in cancer cells, and the accomplice genes that help cancer cells to evade the surveillance of innate and adaptive immunity. So we design the core of the system to simultaneously express KRAS, CD47 and PD-L1 siRNAs. Once they were delivered, the Kras siRNA would inhibit cancer cell proliferation, the CD47 and PD-L1 siRNAs would restore the innate and adaptive immunity to cancer cells. Together, the siRNAs will remove the defense of cancer cells and eliminate them eventually.
2.2 Targeting system
iRGD peptide was reported to specifically bind to the highly expressed αv integrin in tumor cells. We chimerize the iRGD peptide segment to the N-terminus of Lamp2b, a protein expressed on exosome membrane. The fused protein will be incorporated to the exosome memberane and display the iRGD guiding tag on the outside. This design will enhance our targeting ability and increase the delivery efficiency and therapeutic efficacy.
2.3 Exosome booster system
To further enhance the efficiency of exosome delivery, we designed an exosome booster system. KIBRA has been reported to stabilize Rab27 by inhibiting the degradation of Rab27a by proteasomes, which has been shown to promote the release of exosomes in vivo and in vitro. By overexpressing KIBRA in siRNA producing cells, we can speed up the production of therapeutic exosomes and boost the whole therapeutic systems.[5]
2.4 Tet-On System
The Tet-on system consists of a regulatory expression module and a response expression module. The regulatory expression module contains a human cytomegalovirus early promoter (PhCMV) and a reverse tetracycline transcriptional activator (rtTA). Response expression module consists of Tet-responsive element (TRE) and minimal CMV promoter (PminCMV) In the absence of Dox, rtTA cannot bind to TRE, resulting in suppressed gene expression; in the presence of Dox, rtTA can bind to TRE, which in turn activates PminCMV to enable gene expression.[6][7]
2.5 Cre-loxP system
The Cre recombinase recognizes reverse repeats at both ends of the loxP site The DNA sequence between the two loxP sites was cut off by Cre recombinase and quickly link by DNA ligase. The result of recombination depends on the location and direction of loxP sites. In our design, two loxP sites are located on the same DNA strand in the opposite direction, the Cre recombinase will inverse the sequence between loxP.[7]
3. Expected Results
When the systems are simultaneously introduced into liver by lentivirus, the targeting delivery of therapeutic exosomal siRNA will be ready to go. In the presence of doxycycline, the Tet-On system will be specifically expressed in hepatocytes and turn on the expression of three siRNAs and the targeting peptide iRGD, which packaged into therapeutic exosomes and release into circulation. On the other hand, the expression of Cre protein under the control of Tet-On system activates the Cre-loxP system and initiates the expression of exosome booster system. Driven by the exosome booster system, each cell will become an independent factory, with four production lines working together to assemble and secrete the therapeutic exosomes into the circulatory system. Those exosomes will specifically target distal NSCLC cells through iRGD, then release the contained KRAS-siRNA, PD-L1-siRNA and CD47-siRNA, which can kill cancer cells by inhibiting cancer proliferation and metastasis and activating innate and adaptive immunity.
Reference
[1] Moore AR, Rosenberg SC, McCormick F, Malek S. RAS-targeted therapies: is the undruggable drugged?. Nat Rev Drug Discov. 2020;19(8):533-552.
[2] Aguilar EJ, Ricciuti B, Gainor JF, et al. Outcomes to first-line pembrolizumab in patients with non-small-cell lung cancer and very high PD-L1 expression. Ann Oncol. 2019;30(10):1653-1659.
[3] Weiskopf K, Jahchan NS, Schnorr PJ, et al. CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer. J Clin Invest. 2016;126(7):2610-2620.
[4] Tian Y, Li S, Song J, et al. A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials. 2014;35(7):2383-2390.
[5] Song L, Tang S, Han X, et al. KIBRA controls exosome secretion via inhibiting the proteasomal degradation of Rab27a. Nat Commun. 2019;10(1):1639. Published 2019 Apr 9.
[6] https://2013.igem.org/Team:SYSU-China/Project/Design
[7] Hasan MT, Althammer F, Silva da Gouveia M, et al. A Fear Memory Engram and Its Plasticity in the Hypothalamic Oxytocin System. Neuron. 2019 Jul 3;103(1):133-146.e8..