In the Design section, we introduced the conception of the whole project. In order to realize our idea, we rationally designed hierarchical genetic circuits that allows the verification of each parts and the free combination of different functional modules. Then we confirm the expression and function of each part in vitro and in vivo, followed by the confirmation of each system. After that, we will package the whole system with into a lentivirus delivery system, of which the pathogenic genes have been deleted. The lentivirus can effectively infect human cells and be reprogrammed into a exosome production unit by continuous expression of the circuits. Eventually, we will test the therapeutic effect of our strategy in KRAS mutation driven lung cancer mice model.
The design of each module is shown below.
According to our plan, this year we finished the verification of RNAi part, Exosome Booster part and Targeting part. Next year, we will test the in vivo efficacy and therapeutic effect of our strategy.
Targeting Part
For the design of the targeting part, a sequence encoding a tissue-targeting tag was fused to the N-terminus of Lamp2b (a canonical exosome membrane protein). Then the fused protein was inserted downstream of the CMV promoter. Upon translation, this tag should be anchored to the exosome surface via Lamp2b, thus guiding the delivery of the exosomal cargo to the desired tissue. In our project, we chose the well-known homing peptide iRGD (CRGDKGPDC) as the targeting tag, which will penetrate normal tissues and guide the exosomes binding to integrins specifically expressed on tumor vasculature. We confirmed the expression of iRGD-Lamp2b in HEK293T cells by RT-qPCR, which proved that our parts are effective. The targeting of iRGD-Lamp2b to lung cancer cells has been fully studied, so we have sufficient reasons to believe that it can achieve a good target.
Figure 1. iRGD-Lamp2b is expressed in HEK293T cells
RNAi Part
We first designed the expression part of each siRNA. Then, we tandemly linked the siRNA expression cassettes to form the RNAi part. This design enables the co-expression of three different siRNAs for the simultaneous downregulation of multiple therapeutic targets, which will inhibit the proliferation of cancer cells (KRAS) and remove the disguise cancer use to evade the immune system (PD-L1 and CD47) at the same time.
siRNA expression
The siRNA expression cassettes were inspired by the natural precursor of miRNA expression, and we replaced the miRNA sequence with siRNA sequence (see Engineering Section). First, we designed the individual cassettes that express KRAS (siRK), PD-L1 (siRP) and (siRC) separately. Then the cassettes were inserted downstream of the iRGD targeting part (previously reported) and detected the expression of these siRNAs through RT-qPCR. A significant amount of KRAS, PD-L1 and CD47 siRNAs were detected in HEK293T cells transfected with the correspondent cassettes, while no siRNA expression were detected in the negative control (NC) group transfected with cassette expressing scramble sequence. These results indicate that the design of cassettes can correctly express siRNAs together with the targeting part. Then we tandemly link the three cassettes together to construct the iRGD-siRP/C/K module, which was tested for the co-expression efficiency of siRNAs through similar experiments. The results indicated that the iRGD-siRP/C/K module not only successfully expressed the siRNAs simultaneously, but the tandem linking of three siRNA cassettes did not undermine the efficiency of siRNA expression.
Figure 2. Test of siRNA cassettes and RNAi parts in vitro
siRNA secretion
Next, we tried to find out whether these siRNAs can be secreted through the natural exosome secretion system of mammalian cells. We harvested exosomes from HEK293T cell medium after they were transfected with the siRNA cassettes (siRK, siRP or siRC) or the iRGD-siRP/C/K RNAi part. Each kind of siRNA was detected in the exosomes from the correspondent cassette group , and all of the siRNAs were found in the RNAi part group. Also, there is no significant difference in secreted siRNA level generated by iRGD-siRP/C/K RNAi part and each individual siRNA cassette. These results revealed that the siRNAs produced by the RNAi part can be readily packaged into exosomes and secreted into the extracellular environment.
Figure 3. siRNA is packaged in exosomes.
So far, we can confirm that the iRGD-siRP/C/K RNAi part can produce and secrete siRNAs as designed.
Absolute quantification
In order to ensure the content of siRNA in exosomes, siRNA standard was used as the absolute quantification.
Figure 4. Absolute quantification of intracellular siRNAs and exosomal siRNAs
Table1. siRNA Concentration in exosome and cell
| Intracellular siRNA Amount (10-4 pM) |
Exosome siRNA Amount (10-4 pM) |
Exosome Content (mg) |
|
|---|---|---|---|
| KRAS siRNA | 913.8 | 28.48 | 12.66 |
| PD-L1 siRNA | 3.946 | 0.4370 | 27.33 |
| CD47 siRNA | 5.710 | 0.2544 | 24.13 |
According to the absolute quantification of siRNA, we found that the concentrations of all three kinds of siRNA are appropriate. Meanwhile, the concentration of siRNA in exosomes is about 5% of the siRNA in cells.
Exosome booster part
As previously reported, CMV-hSDC4-STEAP3-NadB can strongly promote exosome release. To develop the more efficient booster part for our strategy, we designed another two parts (CMV-KIBRA and CMV-nSMase2) to find out the most efficient design for our project. We tested all the three designs in vitro. The expression of each mRNA was confirmed by RT-qPCR after transfected to HEK293T cells.
Figure 4. KIBRA, NadB, nSMase2 mRNA relative expression in HEK293T cell (vs GADPH)
To further verify the expression of KIBRA and nSMase2 at protein level, we used Western Blotting experiment to prove that KIBRA and nSMase2 was indeed overexpressed in HEK293T cells.
Figure 5. (A)Western Blotting result shows that nSMase2 is overexpressed in HEK293T cell.
(B) Western Blotting result shows that KIBRA is overexpressed in HEK293T cell.
Figure 6. Total amounts of exosomes (shown as total protein) secreted by HEK293 cells with the introduction of nSMase2, KIBRA and hSDC4-STEAP3-NadB.
After confirming the correct expression of these three genes, we further compared their ability to promote exosome secretion. To simplify the process, we first detected the total concentration of the exosomes produced by cells transfected each part. The result indicated that overexpression of KIBRA generates the highest amount of exosomes among these three groups. So we chose KIBRA as the candidate for further characterization.