CO-BERA Live Biotherapeutic for Asthma

Current treatment options, such as corticosteroids and bronchodilators, are often broad in action, leading to systemic side effects and incomplete control of inflammation, such as corticosteroids and bronchodilators.

A hallmark of asthma attacks is the simultaneous occurrence of two signals in the lungs:

1. Acidification in inflamed tissue.

2. Oxidative stress H2O2.

Our solution is CO-BERA, a live biotherapeutic using Lactobacillus plantarum, an engineered probiotic that acts as a smart sensor and therapeutic delivery system.

Inside Lactobacillus plantarum we integrate five genetic devices for delivering CO-BERA successfully:

1. pH sensitive LacR expression device+ Rep protein expression device: Detects low pH and acts as a gate to turn OFF a repressor protein.

2. CO-BERA expression device: Detects oxidative stress, allowing therapeutic production only during inflammation.

3. MV-RNA Loader (DUF-L7Ae) expressing device: Packages therapeutic siRNA into membrane vesicles.

4. H2O2 and PH sensitive endosomal escape device: Helps CO-BERA escape the endosome inside lung cells.

5. Safety Kill Switch: Prevents engineered bacteria from reaching outside the body and blood.

After that , we validate our project in lab by Therapeutic action validation devices in human cell line (lab): CO-BERA siRNA degrades TSLP mRNA, reducing inflammation.

LacR expression device, Rep protein expression device.

This device serves as the master of our therapeutic system. At neutral pH there is a constitutively production of repressor protein (Rep) by Rep protein expression device that keeps our therapeutic gene (CO-BERA) suppressed. This ensures that CO-BERA never accidentally produced and it expressed only when the sensory device (LacR expression device) detects acidity then it sends a signal to suppress Rep protein production result in CO-BERA expression under control of PkatA promoter that activated in presence of H2O2.

CO-BERA expression in lactobacillus conditioned By H2O2 in design

This device represents the heart of our project. We have engineered it as a dual-key regulatory system to ensure absolute precision, so that production of our CO-BERA is only expressed when two conditions of an asthma attack happen simultaneously H2O2 and acidic pH as the following: at acidic environment the first key is removed (Rep protein) by LacR then the second key is removed when PkatA promoter sense H2O2, driving the expression of our therapeutic CO-BERA to target and degrade TSLP which is the key cytokines of inflammation in severe asthma.

MV-RNA Loader (DUF-L7Ae) expressing device

Every powerful therapy needs a special delivery system as a result we engendered Our MV-RNA Loader which is the composite part of this process acts as delivery system for our therapeutic cargo using a specialized fusion protein that acts as both an anchor and a grappling hook as the following: its DUF domain firmly embeds within the bacterial membrane(transmembrane protein), while the L7Ae domain acts as a high-affinity hand, which specifically recognizing our CO-BERA-CD/Box then anchor the CO-BERA to the membrane confirming every budding membrane vesicle is preloaded with the our therapeutic cargo.

H2O2 and PH sensitive endosomal escape device

Delivering a therapeutic CO-BERA into a human cell is only half the battle. The second half, we put into our consideration, which is escaping from its cellular prison when membrane vesicles are taken up by human cell then fused within an endosome that destroys our therapeutic cargo. Consequently, we designed our endosomal escape device to express a mutated form of Listeriolysin O (LLO-L461T) under regulation of H2O2 senstive promoter (PkatA). This mutant LLO retains pore-forming activity, but becomes active at a higher, near-neutral pH. This means it will trigger pore formation earlier, before RNA cargo is degraded, allowing rapid release of siRNA into the cytosol, where it can finally perform its function.

All of these devices work together as the following:

Asthma inflammation signalS (H₂O₂ release,acidic environment): pH sensitive LacR expression device senses acidic expresses LacR which inhibit the repressor of CO-BERA expression (REP), causing CO-BERA expression by exposing PkatA promoter sensitive to H2O2H and in parallel endosomal escape device express LLO.

Loading system: The MV-RNA Loader, which is expressed constructively, and anchors CO-BERA to the bacterial membrane that will form MVs. Then these MVs carry the MV-RNA Loader-CO-BERA complex and LLO to lung epithelial.

Endosomal escape:human cells uptake the MVs into acidic endosomes, then acidic pH activates LLO → pores form → MVs escape to the cytoplasm, resulting in CO-BERA siRNA is released into.

Lab system

Therapeutic action validation device in human cell line

1. TSLP and GFP co-expression device in human cell line.

2. CO-BERA expression device in human cell line.( Therapeutic Action Device (CO-BERA siRNA)

Description

We integrate these devices for testing therapeutic CO-BERA that target TSLP mRNA in the lab. We simulate a human cell environment. After that, we co-express CO-BERA with mRNA that is designed to carry TSLP, T2A, and GFP CDS. On the other hand, we take the same steps in other cells, which simulate human cells. In contrast, we expressed only TSLP, T2A, and GFP. The results show the cells without therapeutic CO-BERA emit green fluorescence, while cells with therapeutic CO-BERA show no green fluorescence. This lab test confirmed that therapeutic CO-BERA is effective in degrading TSLP.

Basic parts

New composite parts

Improved parts

To summarize our improved part