In order to effectively complete our design, we will be divided into four parts to describe switch part, targeting part, RNAi part and promoting exosome release part.
Switch Part
Our project consists of two switch systems :Tet-On system and Cre-loxP system.
The tetracycline on (Tet-on) system, which is also known as the rtTA-dependent system, has been widely-used in genetic studies to enable the conditional regulation of gene expression in a wide variety of cells, tissue cultures, and transgenic animals[1].
It is composed of two elements: the ligand-dependent transactivator tetracycline rtTA as the effector and a TetO-cytomegalovirus (TetO-CMV) minimal promoter cassette regulating the expression of the transgene as the responder. In the presence of tetracycline, or one of its analogs like doxycycline (Dox) or tetracycline(Tet) , rtTA binds to TetO-sequence and activates the transcription of target genes[2-3].
Researchers have combined the Tet-on inducible expression system with tissue-specific promoters (e.g., albumin promoter for liver-specific expression[4]) to achieve both temporal and spatial control of the expression of genes-of-interest for functional studies. All these prove that the Tet-On system integrates Albumin promoters into the expression system.
The expression of Cre recombinase is doxycycline activated because it is controlled by the Tet-on system and has also been shown to be feasible and effective. Therefore, we believe that the combination of the Tet-On system with the Cre-loxP system will be effective and useful[5].
Target Part
We use iRGD-Lamp2b as our target part. When the cells are transfected into the plasmid they will express Lamp2b in fusion with the tumor-penetrating iRGD peptide (CRGDKGPDC) which has a high affinity to integrin αvβ3. We can use iRGD-Lamp2b to complete the docking between exosomes and tumor cells and complete targeted therapy[6].
RNAi Part
In order to treat the non-small cell lung cancer caused by KRAS, we designed siRNA for KRAS, and designed siRNA and for PD-L1 and CD47. The tumor cells will express the PD-L1 signal.
The binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM). Tumor cells escape macrophage phagocytosis by overexpressing CD47. At the same time, siRNA targeting CD47 was produced, and mRNA expression was targeted to be reduced. This allows macrophages to avoid the effects of CD47 and attack tumor cells.
By simultaneously expressing siRNA targeted at KRAS, PD-L1 and CD47, we not only inhibited the proliferation of tumor cells but also enhanced the immune system's role in tumor cell clearance, so as to eliminate cancer.
Our experiments have proved that three siRNA can be expressed in HEK293T cells, and exosomes contain a certain amount of siRNA. Moreover, The expression of KRAS siRNA was verified by RT-qPCR assay to reduce the expression of KRAS.
Exosome Booster Part
In order to ensure the effectiveness and sustainability of the system, after comparing nSMase2, hSDC4-STEAP3-NadB and KIBRA, we selected KIBRA as the part to promote exosome release. 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[7].
By promoting exosome release, we ensured that cells produced enough exosomes containing siRNA to enhance efficacy.
To our Result: https://2020.igem.org/Team:NJU-China/Results
Reference:
[1] Zhou, et al,.Optimization of the Tet-On system for regulated gene expression through viral evolution.Gene Ther. 2006, 13(19):1382-90.
[2] Burger, et al., Generation of a novel rtTA transgenic mouse to induce time-controlled, tissue-specific alterations in Pax2-expressing cells. Genesis. 2011, 49(10):797-802.
[3] Thiem, et al., Inducible gene modification in the gastric epithelium of Tff1-CreERT2, Tff2-rtTA, Tff3-luc mice. Genesis. 2016, 54(12):626-635.
[4] Gandhi, et al., Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice. Gastroenterology. 2015 148(2):379-391.
[5] Jiang, et al,. A Tet-on and Cre-loxP Based Genetic Engineering System for Convenient Recycling of Selection Markers in Penicillium oxalicum. Frontiers in Microbiology. 2016, 7:485.
[6] Kazuki, et al., Tissue-penetrating delivery of compounds and nanoparticles into tumors. Cancer Cell, 2009.
[7]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.