Design At the beginning of our project, we first aimed to silence Interleukin-13 (IL-13) because it is one of the key cytokines and has an important role in the asthma cascade. Blocking IL-13 was expected to reduce airway hyperresponsiveness, mucus production, and remodeling, making it a rational therapeutic target (1,2).

Build Our first design strategy therefore, focused on engineering an RNA therapeutic against IL-13, with the goal of preventing its downstream inflammatory effects in severe asthma.

Test Through an extended literature review, we discovered important limitations:

• IL-13 primarily regulates type-2 eosinophilic asthma, but it does not adequately cover other clinically important phenotypes, such as non-type 2 (neutrophilic) asthma (1).
• IL-13 silencing alone would not effectively tackle corticosteroid-resistant asthma (2).

Learn Upon further analysis and after meeting with Dr/ Ahmed Beshir , we realized that targeting IL-13 offered limited clinical benefit and might exclude patients with non-type-2 asthma. As a result, we redirected our attention to Thymic Stromal Lymphopoietin (TSLP), an upstream cytokine that drives both type-2 and non-type-2 inflammatory pathways and is strongly implicated in corticosteroid resistance (3,4). By shifting our therapeutic target from IL-13 to TSLP, we enhanced both the clinical relevance and the potential impact of our project.

Design After choosing TSLP as our molecular target, we then started to think about how to silence it and which level of the central dogma we should target. Our first thought was to use Antisense Oligonucleotides (ASOs) to turn down its activity at the post transcriptional level. We designed ASOs to target and bind specific mRNA sequences (TSLP mRNA) and inhibit its expression (1).

Build Our strategy was based on utilizing ASOs as the primary therapeutic element within engineered vectors. The rationale was that ASOs, delivered via synthetic liposomal vesicles, would hybridize with TSLP mRNA in host cells and suppress its expression (2).

Test After Finishing our model and upon feedback from Dr/ Mohammed Hussein that highlighted several substantial limitations:

• ASOs require significant chemical modifications, such as alterations to their backbone or sugar moieties, to remain stable and effective in vivo (3,4).
• These necessary modifications are not naturally biosynthesized, making them difficult to produce at scale (5).

In addition of the result of our modeling As shown in this figure:
Bar graph comparing TSLP knockdown efficiency across four conditions: untreated (Natural), Antisense Oligonucleotides (ASO), BEBA, and CO-BEBA. ASO treatment achieved 79.0% knockdown, outperforming the Natural baseline (11.6%) but falling short of BEBA (82.5%) and CO-BEBA (98.7%). These results, combined with scalability and stability concerns, led to the strategic pivot away from ASO-based silencing toward biologically encoded delivery platforms.
Given these challenges, this strategy proved impractical and necessitated reevaluation.

Learn From these findings, we concluded that ASOs were not feasible for our project. So we decided to search for another Silencing system. As a result of discussions with Dr/ Raghda, we therefore shifted toward using CO-BERA, a dual-function siRNA system. Unlike ASOs, siRNAs can exploit the natural RNAi pathway without requiring chemical modifications for activity, making them more suitable for biological production. and more reliable as a therapeutic component (6–8).
Advances in RNA bioengineering (6,7) support CO-BERA as a scalable and effective solution.

Design Following the selection of CO-BERA as our therapeutic agent, we turned our attention to the delivery system. Our initial plan was to use liposomes as carriers. Liposomes are widely used for drug delivery because they can encapsulate nucleic acids and fuse with host cell membranes, enabling intracellular delivery (1).

Build We created a liposome to be our delivery vehicle, where CO-BERA molecules would be packaged and administered directly to the lungs. The rationale was that liposomes might offer a controlled release system, enabling local delivery to airway epithelial cells (2).

Test However, a deeper evaluation and more literature review showed significant Limitations:

• Conventional liposomal formulations tend to be cleared rapidly or degraded at the site of administration; specialized approaches (e.g., PEGylation or LNP formulations) can be used to improve stability but add to formulation complexity and cost (3).
• They needed repeated administration, particularly for chronic respiratory diseases where mucociliary clearance and local degradation reduce residence time (4).
• Large-scale, stable encapsulation of RNA molecules into liposomes remains technically a problem and costly (5).

Learn These limitations made liposomes unsuitable as our delivery platform. We therefore shifted toward using Corynebacterium as a living chassis. Unlike liposomes, bacteria can act as continuous “factories,” capable of producing therapeutic agents. This approach promised longer persistence, reduced dosing frequency, and adaptability compared to non-living delivery systems (6).

Design After narrowing down our delivery system, we considered Corynebacterium as the chassis for therapeutic production. It is a Gram-positive bacterium capable of secreting vesicles and can be engineered for nucleic acid expression (1).

Build We explored Corynebacterium as the microbial “factory” that would continuously release our therapeutic CO-BERA into the lung environment. Its non-pathogenic strains have been used in biotechnology, making it a candidate worth evaluating (1).

Test Further research, however, highlighted limitations:

• Corynebacterium lacks strong evidence of direct clinical benefit in the respiratory tract.
• It has limited immunomodulatory activity, providing no additional therapeutic effect beyond acting as a carrier.
• Its colonization dynamics in the lung are less characterized compared to probiotic organisms (2).

Learn After more research about probiotic organisms in the respiratory tract, we decided to use Lactobacillus plantarum, a probiotic species naturally associated with the gut–lung axis. Unlike Corynebacterium, L. plantarum not only used as a delivery chassis but also provides intrinsic therapeutic benefits, including:

• Anti-inflammatory effects through modulation of NF-κB and MAPK pathways (3).
• Reduction of IgE and pro-inflammatory cytokines, key markers of asthma (4).
• Enhancement of host antioxidant defense mechanisms, improving respiratory function (5).

Thus, Lactobacillus plantarum provides a dual-action advantage: it acts both as a chassis for CO-BERA delivery and as a therapeutic agent due to its anti-inflammatory function This made it the ideal choice for our final system.

Design Our initial approach placed CO-BERA under the control of the KatA promoter, which is activated by oxidative stress (H₂O₂). This strategy was selected based on multiple studies reporting that KatA is strongly induced during oxidative bursts in Lactobacillus and other Gram-positive bacteria. We expected this design to provide disease-specific activation in inflamed airways.

Build In our system, we implemented an inflammation-dependent promoter system for CO-BERA expression. Specifically, we chose H₂O₂-sensitive promoters (KatA promoter) that respond to disease hallmarks, as elevated oxidative stress (H₂O₂) changes in inflamed airways. The KatA promoter, which is repressed under normal conditions by PerR but induced upon the presence of reactive oxygen species, was chosen as one of the key switches.

Test However, both our modeling and literature reviews revealed that pKatA shows basal leakage under non-stress conditions. Specifically, PerR repression was not absolute, meaning residual transcription could occur even in healthy environments. This leakage risked unintended CO-BERA expression, raising concerns for off-target activity.
shown Conditioning model
Time-course analysis of pKAT mRNA expression (mM) under varying concentrations of hydrogen peroxide (H₂O₂), ranging from basal (no H₂O₂) to 0.15 mM. Expression levels increased proportionally with H₂O₂ concentration, plateauing after ~5 hours. The basal expression level (13.36 mM) accounted for 40.08% of total activation, indicating basal leakiness. These data validate pKAT as a tunable oxidative stress–responsive promoter suitable but show issue for conditional gene expression systems.

Learn From this, we concluded that H₂O₂ alone was not sufficient for precise therapeutic control. A more stringent dual-conditioning system was needed to minimize off-target effects.

Rebuild We engineered an additional pH-sensitive layer of repression:

• Inserted a Rep operator downstream of pKatA.
• The Rep repressor was placed under the constitutive p32 promoter.
• To introduce pH sensitivity, we integrated a Lac operator within p32.
• In inflamed lungs (pH < 6.9), LacR is activated through the PH sensitive promoter (P170-CP25), and then binds the Lac operator, and inhibits p32. This reduces Rep levels, relieving suppression of KatA.

Thus, CO-BERA expression became dependent on two concurrent inflammatory signals: elevated H₂O₂ and acidic as mentioned in Approach design page.

Re-Test Based on our logic- AND Gate model and supportive literature reports on dual-input promoters, the reconstructed circuit showed:

• Markedly reduced basal leakage under neutral pH / low oxidative stress.
• Robust expression under combined acidic pH and oxidative stress, mimicking asthmatic lung conditions.

Description of Dual-Input AND Gate Circuit Performance
The dual-input AND-gated genetic circuit was reconstituted for CO-BERA expression under the combined stimuli of pH and oxidative stress. The neutral pH and low oxidative stress conditions representative of a healthy lung showed a markedly low basal leakage of the genetic circuit, thus effectively repressing the expression and thereby preventing any unwanted activation. Contrarily, elevated H₂O₂ and acidic pH are the simultaneous conditions set up by inflamed asthmatic airways, and hence the circuit was fully turned on in transcription, with CO-BERA mRNA level spikes and settling to around 20 mM. Such a conditional response assures the greatest fidelity of the AND gate logic to further limit therapeutic gene expression to a disease-specific microenvironment. These results are in agreement with dual-input promoter literature and thus establish the concept of the circuit for pulmonary inflammation-targeted intervention.

Learn This iteration established a dual-gated, highly specific expression system. By integrating insights from both modeling and literature, we eliminated the leakage problem while maintaining strong therapeutic activation under disease-relevant conditions.

Design Although our CO-BERA system was successfully loaded and conditionally expressed, there was one significant limitation: therapeutic RNAs delivered through bacterial membrane vesicles face endosomal trapping once inside host cells. Literature shows that only 0.3% of RNA cargos naturally escape endosomes, severely limiting therapeutic efficacy (1). To overcome this barrier, we sought a strategy for enhancing endosomal escape.

Build After searching, we found Listeriolysin O (LLO), the pore-forming protein from Listeria monocytogenes that has the ability to disrupt endosomal membranes and help in endosomal escape (2).

Test Several studies mentioned the risk of using wild LLO due to its pathogenicity, as LLO is one of the main factors enabling Listeria monocytogenes to escape the phagosome and establish infection inside host cells(3,4).
So using the wild protein directly could mimic pathogen-like activity, raising biosafety concerns.

Learn So we decided to use a less virulent version of this protein that is less harmful and useful in therapy.

Rebuild To ensure safety and avoid pathogenic effects, we designed and incorporated a mutated LLO. To be safe and prevent pathogenicity,We used an engineered LLO variant shown in the literature to have strongly reduced pathogenic activity while retaining pH-dependent pore formation compared to wild-type LLO (2,3). This allowed us to preserve its endosome-disrupting function while eliminating potential pathogenesis.

Re-Test Our analysis of existing literature confirmed that engineered LLO mutants retain the ability to mediate pH-dependent pore formation in endosomes, without the risks associated with the wild-type protein (3). In silico protein modeling validated the lower toxicity and sustained structural stability of the chosen LLO variant.

Learn With the integration of mutated LLO, our system gained a powerful mechanism for endosomal escape, ensuring that a much higher fraction of CO-BERA reaches the cytoplasm, where it can effectively silence target genes (3,4). This step is important for enhancing the efficiency and therapeutic potential of our probiotic-based delivery platform.

Design Although Lactobacillus plantarum is considered safe as a probiotic and naturally functions along the lung–GIT axis (1), our team identified a potential biosafety concern: the possibility of bacterial escape beyond the intended environment. Uncontrolled persistence of engineered strains outside the lung could pose risks to patients and the ecosystem. To mitigate this, we sought a genetic containment mechanism.

Build We added a Toxin–Antitoxin (TA) system into our design. In this system, the antitoxin is continuously expressed within the lung environment, which neutralises each other, ensuring bacterial survival and therapeutic function. However, if bacteria escape outside the lung and lose access to required regulatory signals, the antitoxin is degraded, while the stable toxin remains active—leading to self-elimination of the escaped bacteria (2,3).

Test We reviewed successful applications of TA systems in synthetic biology, where they act as biological kill-switches (3,4). Modeling confirmed that such a system could maintain bacterial survival in the lung while providing kill-switches against uncontrolled dissemination.

Learn By integrating the TA system, we significantly advanced the biosafety and controllability of our probiotic platform. This ensured not only therapeutic efficacy but also compliance with biocontainment standards(5).

Design After successfully designing our therapeutic RNA scaffold (CO-BERA), with further search, we noticed that only a small fraction of the RNA could be efficiently loaded into bacterial membrane vesicles. To overcome this limitation, we looked for strategies that would enhance RNA localization to vesicles.

Build We proposed to use RNA-binding proteins (RBPs) to guide CO-BERA closer to the vesicle formation sites. Literature pointed to the Cold Shock Protein (CSP) family in Lactobacillus, which are naturally present and capable of binding RNA.

Test Our design relied on CSP’s ability to bind RNA and thus enrich CO-BERA molecules near vesicle membranes, increasing loading efficiency.

Learn Initial analysis suggested CSP could indeed bind RNA, but its role in vesicle loading was uncertain. We decided to search more about whether CSP could serve as a localization factor for packaging RNA into vesicles.

Design We first considered Cold Shock Proteins (CSPs) as RNA-binding proteins (RBPs) for loading CO-BERA into membrane vesicles. However, literature revealed that CSPs are cytoplasmic rather than membrane-bound, meaning they would not ensure accurate localization of RNA into vesicles. We then searched for RBPs naturally linked to transmembrane proteins (TMPs) in Lactobacillus plantarum, but found no published evidence of such systems in Gram-positive bacteria. This pushed us to pursue an engineered solution.

Build Our first design strategy was to create a fusion protein consisting of the membrane-anchored foldase PrsA linked to L7Ae (a well-characterized RBP) through a flexible linker. The idea was that PrsA would anchor the construct to the membrane while L7Ae bound the RNA cargo.

Test The PrsA–linker–L7Ae construct was modeled using SWISS-Model, CHARMM-GUI, and tested through molecular dynamics simulations in GROMACS. These simulations demonstrated unstable orientation and poor anchoring in the membrane, suggesting that PrsA was not a suitable choice for this application.

Learn From this outcome, we learned that the choice of TMP is crucial, as not all anchors provide structural stability or proper membrane integration when fused to RBPs.

Rebuild We selected DUF4811, a transmembrane protein found in MV of Lactobacillus and designed DUF4811–linker–L7Ae fusion constructs for testing.

Re-Test Computational modeling with SWISS-Model and CHARMM-GUI, followed by GROMACS molecular dynamics simulations, confirmed stable membrane orientation.

Learn WThis established DUF4811–L7Ae as a stable and reliable RNA loading system, supporting efficient localization of RNA to vesicles.

Design Initially, we aimed to deliver our engineered Lactobacillus plantarum using aerosolization. This approach is widely used for liquid-based drugs and was expected to ensure rapid and broad dispersal of our bacteria into the lungs (1,2).

Build We considered aerosol devices that generate fine droplets capable of reaching the lower respiratory tract, making them a plausible option for live biotherapeutic delivery (2).

Test Upon reviewing the literature and experimental data on bacterial delivery methods, several challenges with aerosolization emerged (3,4):

• Shear stress during nebulization reduces bacterial viability.
• Short residence time in the lung due to mucociliary clearance.
• Higher risk of contamination and reduced stability during storage.

Learn From this outcome, we learned that the choice of TMP is crucial, as not all anchors provide structural stability or proper membrane integration when fused to RBPs.

• Better preservation of bacterial viability during formulation and storage.
• Longer half-life and stability of the probiotic formulation.
• Greater patient compliance and applicability, as DPIs are widely used in asthma management.

This switch allowed us to design a delivery system that is not only effective but also practical and scalable for patients with asthma.

Design After selecting Dry Powder Inhalers (DPIs) as our delivery platform, we initially chose spray-drying to formulate the bacterial powder. Spray-drying is a fast, continuous, and widely used industrial process for producing dry formulations, making it attractive for scalable therapeutic production

Build We considered spray-drying conditions commonly used for probiotics, where heated air rapidly evaporates the solvent to produce a fine dry powder suitable for inhalation.

Test Further research and our modeling revealed significant drawbacks of spray-drying for our application:

• High temperature exposure reduces probiotic viability.
• Rapid dehydration can damage bacterial membranes.
• Poor long-term survival rates limit clinical feasibility.

Bar chart comparing probiotic survival rates following two drying methods: Freeze-Drying and Spray-Drying. Freeze-Drying achieved a higher survival rate (72.7%) compared to Spray-Drying (59.5%), with a noted advantage of +13.2%. These results support the selection of Freeze-Drying for DPI formulation, due to its superior preservation of bacterial viability and reduced process-related risks.

Learn After the result of our modeling and taking into account guidance from Dr/ Ahmed Abdelsabour, we transitioned to freeze-drying (lyophilization) as the preferred method.
As Freeze-drying offers:

• Superior preservation of bacterial viability compared to spray-drying.
• Lower stress on bacterial membranes during processing.
• Compatibility with protective excipients that improve powder stability and dispersion.

This choice ensures that our Lactobacillus plantarum remains viable throughout production, storage, and delivery to the patient’s lungs.

Design

• Plan to amplify and domesticate Fragments 1, 2 and 3 from Twist DNA for use in CO-BERA and loading circuits.
• Designed primers with overhangs for restriction and Golden Gate cloning.

Build

• Resuspended fragments and primers.
• PCR amplification of all fragments.
• Transformation of the TagBFPV2 plasmid into E. coli Jm109 to validate the M13 primer system.

Test

• Gel electrophoresis confirmed the correct amplification of Fragments 1 and 2.
• Fragment 3 gave inconsistent gradient bands.
• Transformation of TagBFPV2 was successful, confirming that M13 primers work.

Learn

• PCR of Fragment 3 required optimization → annealing temperature adjusted to 60 °C.
• Validated workflow for primer use and colony screening.