Enhancing complement system against Leptospira
Design
We have tried different approaches to our inhibitor design and decided on enhancing the human immune system, through the use of MicroRNA (miRNA) mimics, to reduce the effects of self-regulatory miRNA in our body.
Before settling on this current design, we tried using miRNAs to degrade the Leptospira protein. However, after consulting professionals from The University of Hong Kong (HKU), we were told that our original design was not feasible which brought us back to the drawing board.
In order to address this problem, we first looked at reasons of why our own immune system was able to combat leptospira naturally. We discovered that leptospira has many ways of evading the detection by the immune system intentionally.
First, by downgrading itself to conserve energy, it is constantly in an inferior caliber since the membrane attack complexes (MAC) form at a slower rate which would originally puncture the leptospira cell and cause cell lysis. . Looking deeper into the root, we realized that our body uses miRNA, such as hsa-mir-144, hsa-mir-4427, hsa-mir-3646, and hsa-mir-630, to block the messengerRNAs (mRNA) originally used to transport information to our complement system or immune system proteins, such as the Toll-like receptor 2 (TLR2), Complement Factor H (CFH), and C3.
Secondly, different leptospira outer membrane proteins, such as LcpA, LenA and Lsa23, can bind with our body’s complement proteins and inhibit the formation of the MAC.
With our new discoveries, we drafted a new design that focuses on blocking the self-regulatory miRNA with our own miRNA inhibitors.
The self-regulatory miRNAs has a 3’UTR, which essentially works as a guide that gives directions for the miRNA. By attaching onto the 3’UTR, the miRNA will not be able to block the messengerRNAs of our body, and our immune system will be able to combat the leptospirosis infection through a more rapid formation of MAC.
Now that we came up with a feasible design, new issues arise regarding the implementation of our design into the body.
At first, we were all divided against exosomes, viral vectors and nanoparticles as carriers for our design inside a. However, after a meeting with CUHK SBS’s iGEM team, we learnt that exosomes is the most effective form of implementation due to its great biocompatibility with the body and wouldn’t cause immunological rejection unlike the other two options which have low drug-loading efficiency or high toxicity.
Build
With our design in theory, we started working on the 2 constructs based on messengerRNAs C3 and CFH which can be seen below.
Construct based on C3
Construct based on CFH
Construct based on TLR2
Our lab work presents 3 different scenarios, all within an A549 mammalian cell lines, to simulate the environment inside the human body and a bioluminescent vector.
Test
How we know the product works/is successful.
If our lab results align with the following scenarios, this shows that our inhibitors were indeed able to prevent the regulatory miRNAs from blocking the mRNAs.
Firstly, the messenger RNA is in a bioluminescent vector - showing a positive or a highly concentrated result.
Secondly, messenger RNA with micro RNA, showing a less concentrated or negative result.
Thirdly, messenger RNA with micro RNA and its inhibitor- showing a positive result similar to the one in the first scenario.
Based on these 3 findings, the implication that our inhibitors have an upregulatory effect on the body’s mRNAs can be made.
Learn
After performing a luciferase assay we received the following results on our 3 scenarios mentioned above.
From these results, we learnt that we have gained insight for how mammal cell culture and luciferase assay works from HKU staff
In the future we plan to run more trials for luciferase assay. We have run only one test, but instead we should run for 5 more to minimize errors, prevent unfair situations, and also ensure efficiency of data collected. The pharmacokinetic/pharmacodynamic (pk/pd) modeling will be studied for its delivery to the infected organs and our inhibitors can then be evaluated for drug design
