Results
Here we show that unlike the original Cell modules, LoGENEgate totally precludes expression of a model gene in off-target cells. Our findings pave the way for numerous therapeutic applications of LoGENEgate.
Overview
- We design 2 circuits and constructed 3 Parts for each circuit.
- Assembled and demonstrated a functional AND gate gene circuit in Hek293T cells
- Compared the results to find which trans-splicing sites are more effective.
- Compared our system to literature, ours is safer!
Results
- Design and Construction of Circuits
In order to achieve our goal, we first designed and constructed the Trans Splicing (TS)-based AND gate circuits, LoGENEgate.
We have assembled and analyzed two different circuits, each comprising two modules (see below for details), with different intronic sequences designated to induce TS. The first circuit, comprised of TS1 + TS2, include a 'basic' TS site design based on the original intron separating the two mKate2 exons used in cell 20171. The second circuit, TS11 + TS12, contain a TS site design based on a thorough analysis to maximize the efficacy. Each of the modules is regulated by a separate promoter (Figure 2). Module 1 consists of P1 (SSX1p), which regulates the expression of exon 1 of mKate2 as well half of the intron that include a strong donor splice site and linker. Module 2 consists of P2 (H2A1p), which regulates a TS guiding sequence, the acceptor splice site and mKate2 exon 2 (Figure 1).
First circuit:
Second circuit:
Following trans-splicing, the exons are assembled into a mature output mRNA.
In this system, the output protein is expressed only when the promoters regulating both modules are mutually active. However, when only one of the promoters is active or when none of the promoters are active, they cannot produce any functional protein. thus, standing alone, is permanently in state ‘0’.
We defined two different states for this circuit: in state [1,0], module 1 is active, while module 2 is inactive (off); and in state [1,1], both module 1 and module 2 are active (on).
- Expression in HEK293T cells
The data below (Figures 3+4) display the transient transfection of HEK293T cells with plasmids containing LoGENEgate by flow cytometry. Any expression below the negative control (<3.09%) is irrelevant, hence amounts to 0% expression of GOI.
As expected in [1,0] states there is no product, 0% expression, while in [1,1] stats the mean of expression is 24.02%.
For further validation, the experimentwas done twice each on 105 cells, with similar results.
As expected the second 'optimized' circuit, TS11+12, yielded higher levels of expression [31.55% (Figure 4) compare to 17.5% (Figure 3)] then the first 'basic' circuit. However, we note that further optimization is needed.
- Comparing to cell 2017
We next compared the results of LoGENEgate to the results from cell 2017 1 (Figure 5). Although the sponge system is more efficient, with a higher level of expression in state [1,1], LoGENEgate is much safer, with 0% levels of expression compare to 15% in state [1,0].
Conclusion and Future plans
We presented proof-of-concept demonstration that LoGENEgate works!
The protein product of the two-module circuit is only translated when both modules are co-expressed and TS occurs, but is absolutely absent when each of the two modules is expressed alone. Even so our experiments didn't test the promotors themselves. Accordingly, the next step will be to find promotors that are specific to a type of cancer and test the expression of the GOI in healthy cells compare to cancer cells.
Additionally, there is no doubt that several rounds of optimization steps could substantially improve the circuit efficacy, while not impairing its safety at all.
We have patented LoGENEgate and our lab is committed to continue this research and get it to clinical phases.
Reference
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