Team:NJU-China/Design

Project Design

Our project aims to construct a safe and efficient drug delivery system targeting specific oncogene to provide a new strategy of cancer therapy. We chose lung cancer as our disease model due to its substantial morbidity and mortality.

Generally, the project design can be divided into three portions: (1) RNAi module, (2) targeting module, and (3) RNAi module and targeting module assembly.

RNAi module

RNA interference (RNAi) is an emerging technology of gene silencing. It uses siRNA segments to destroy specific mRNA, thereby, shut down the expression of target genes. RNAi has been demonstrated as a novel treatment modality of cancer and we decided to utilize this technology to silence the expression of specific oncogene [1]. KRAS is one of the most commonly mutated oncogenes in lung cancer. The mutation rate of KRAS can be up to 25% in NSCLC (a main type of lung cancer) [2]. Thus, we picked KRAS as our target oncogene. Then we designed anti-KRAS siRNA as a therapeutic agent to degrade KRAS mRNA, therefore, repressing the expression and function of K-ras protein. We used a specialized software developed by team SYSU-software to find the ideal siRNA sequence. This tool also designed pairs of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. When the shRNA plasmids of KRAS are transfected into HEK293 cells, the dsRNA is cleaved into siRNA of KRAS by the enzyme Dicer, and then target KRAS mRNA. However, due to the short half-life period and poor cellular uptake of exogenous siRNA[3], an efficient delivery vehicle is needed to stabilize and enhance the naked small interference molecule's function.

Fig 1. Pre-designed shRNA sequence

Targeting module

Exosomes are natural nano-sized vesicles secreted by numerous cell types [4]. Exosomes derived from cells naturally contain a substantial amount of RNA. Moreover, their membranes endow exosomes with a specific cell tropism, attaching themselves to target cells by a range of surface adhesion proteins and vector ligands. Besides, exosomes are endogenous transporters, so they are unlikely to exert toxicity or immune response [5]. Hence, exosome is regarded as a potentially ideal siRNA delivery tool with advantages in three aspects: RNA-loading, targeting and safety.

Moreover, exosomes can be endowed with the ability of targeting specific disease site through genetic modification of donor cells. In order to obtain exosomes with tumor-targeting ability, we redesigned natural exosomes by installing a tumor-targeting short peptide onto their surface. We chose iRGD, a tumor-penetrating peptide. Some studies have shown that iRGD homes to tumors by strictly binding to αν integrins which are specifically expressed on tumor cells and the endothelium of tumor vessels [6]. Meanwhile, iRGD peptide also own the ability of penetrating tumor tissue against interstitial pressure [7]. When combined with a drug, iRGD peptide can carry the drug deep into the extravascular tumor tissue.

We engineered our chassis, human embryonic kidney 293 (HEK293) cells, to express a fusion protein composed of the exosomal membrane protein Lamp2b and the tumor-targeting short peptide iRGD. Lamp2b (lysosomal-associated membrane protein 2b) is a protein found specifically abundant on the surface of exosomes. By genetically engineering the iRGD peptide to the outer membrane portion of Lamp2b, Lamp2b can bring the iRGD peptide to the surface of exosomes. Then the iRGD peptide can guide exosomes to the specific tumor tissue. Through these modifications, exosomes will be conferred on tumor-targeting ability.

Fig 2. We connected iRGD 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 tumor tissue.

Assembly of the RNAi module and targeting module

During experiment, we transfected siRNA plasmid and target plasmid (coding our engineered fusion protein) into HEK293 cells to obtain exosomes with KRAS siRNA inside and iRGD peptide on their surface membranes. When the modified exosomes are injected into the bloodstream, the iRGD peptide will guide exosomes deep into tumor tissue and the exosomes will specifically recognize tumor cells. Once fused into tumor cells, KRAS siRNA will be released, targeting and destroying KRAS mRNA. In this way, the expression of KRAS will be potently shut down. In theory, the delivery of KRAS siRNA to tumor cells will be achieved, whereas non-specific uptake of KRAS siRNA in other tissues will be avoided. As a consequence, the inhibited expression of KRAS oncogene will repress the proliferation of tumor cells, ultimately presenting therapeutic effects on lung cancer.

Fig3. Schematic diagram of modified exosomes loded with siRNA silencing KRAS oncogene

Reference

[1] Aagaard L, Rossi JJ. RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev. 2007;59(2-3):75-86.

[2] Westra WH, Slebos RJ, Offerhaus GJ, Goodman SN, Evers SG, Kensler TW, Askin FB, Rodenhuis S and Hruban RH. K-ras oncogene activation in lung adenocarcinomas from former smokers. Evidence that K-ras mutations are an early and irreversible event in the development of adenocarcinoma of the lung. Cancer 1993, 72:432-438.

[3] Gavrilov K, Saltzman WM. Therapeutic siRna: Principles, challenges, and strategies. Yale J Biol Med. 2012;85(2):187-200.

[4] A.V. Vlassov, S.Magdaleno, R. Setterquist, R. Conrad, Exosomes. Current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta. 2012;1820(7):940-8.

[5] El Andaloussi S, Lakhal S, Mäger I, Wood MJ. Exosomes for targeted siRNA delivery across biological barriers. Adv Drug Deliv Rev. 2013;65(3):391-7.

[6] Sugahara KN1, Teesalu T, Karmali PP, Kotamraju VR, Agemy L, Girard OM, Hanahan D, Mattrey RF, Ruoslahti E. Tissue-penetrating delivery of

compounds and nanoparticles into tumors. Cancer Cell. 2009;16(6):510-20.

[7] Kazuki N. Sugahara, Tambet Teesalu,Priya Prakash Karmali, Venkata Ramana Kotamraju, Lilach Agemy, Daniel R. Greenwald, Erkki Ruoslahti.

Coadministration of a Tumor-Penetrating Peptide Enhances the Efficacy of Cancer Drugs. Science. 2010;328 (5981), 1031-1035.