Asthma is a chronic inflammatory disease that affects over 300 million individuals in the world and is a significant leading cause of morbidity and a reduction in quality of life. Although there exist available options of inhaled corticosteroids, β2-agonists and biologics like anti-IL-5 or anti-IgE antibodies, there remain numerous patients who are left with refractory symptoms and airway remodeling. Difficult to manage especially are those cases of severe asthma and often with steroid-resistant disease.
From Ideas to Design
In the early stages of our project, each member of the team was involved in the exploration of different technological platforms available for the therapy of Asthma. The suggested concepts represented a wide range of thought such as viral vectors and lipid nanoparticles for siRNA delivery, to antibody-related biologics and small molecules. After several discussions, the team compared these options in terms of safety, specificity, ease of engineering, and long-term applicability.
Throughout the prolonged journey, we experienced numerous challenges that continually shaped our platform. We based upon extensive literature research, the integration of numerous experts, in silico software validation, and ultimately, experimental validation in the laboratory. Each of these hurdles is addressed throughout our DBTL cycles, which resulted in critical modification of our PRESS
References
Rabe APJ, Loke WJ, Gurjar K, Brackley A, Lucero-Prisno III D, et al. Global Burden of Asthma, and Its Impact on Specific Subgroups: Nasal Polyps, Allergic Rhinitis, Severe Asthma, Eosinophilic Asthma. J Asthma Allergy. 2023;16:1097-1113. doi:10.2147/JAA.S41814519.
Mahesh PA, et al. Molecular mechanisms of steroid-resistant asthma. Explor Asthma Allergy. 2023;1:174-185. doi:10.37349/eaa.2023.00018.
Global, regional, and national prevalence of asthma in 2019: a systematic analysis and modelling study. J Glob Health. 2022;12:04009. DOI 10.7189/jogh.12.04052.
Iteration 1
Choosing the Therapeutic Target
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DesignAt 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).
BuildOur 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.
References
Dowdy SF. Endosomal escape of RNA therapeutics: How do we solve this rate-limiting problem? RNA. 2023 Apr;29(4):396-401. doi: 10.1261/rna.079507.122. Epub 2023 Jan 20. PMID: 36669888; PMCID: PMC10019367
Corren J, Lemanske RF Jr, Hanania NA, Korenblat PE, Parsey MV, Arron JR, et al. Lebrikizumab treatment in adults with asthma. N Engl J Med. 2011;365(12):1088–98. doi:10.1056/NEJMoa1106469
Gauvreau GM, O'Byrne PM, Boulet LP, Wang Y, Cockcroft D, Bigler J, et al. Effects of an anti-TSLP antibody on allergen-induced asthmatic responses. N Engl J Med. 2014;370(22):2102–10. doi:10.1056/NEJMoa1402895
Menzies-Gow A, Corren J, Bourdin A, Chupp G, Israel E, Wechsler ME, et al. Tezepelumab in Adults and Adolescents with Severe, Uncontrolled Asthma. N Engl J Med. 2021;384(19):1800–9. doi:10.1056/NEJMoa2034975
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Iteration 2
Selecting the Silencing Technology
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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).
BuildOur 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 to the result of our modeling (As shown in this fig. ), the Bar graph below compares TSLP knockdown efficiency across four conditions: untreated (Natural), Antisense Oligonucleotides (ASO), BERA, and CO-BERA. ASO treatment achieved 70.0% knockdown, outperforming the Natural baseline (15%) but falling short of BERA (82.5%) and CO-BERA (99.7%). 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 shifted toward using CO-BERA which has 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.
References
Holgate ST. Pathogenesis of asthma. Clin Exp Allergy. 2008;38(6):872–97.
Barnes PJ. Targeting cytokines to treat asthma and chronic obstructive pulmonary disease. Nat Rev Immunol. 2018;18(7):454–66.
Østergaard ME, Southwell AL, Kordasiewicz HB, Watt AT, Skotte NH, Doty CN, et al. Chemical modification of PS-ASO therapeutics reduces cellular protein binding and improves the therapeutic index. Nat Biotechnol. 2020;38(12):1431–9.
Wang F, Zuroske T, Watts JK. RNA therapeutics on the rise. Nat Rev Drug Discov. 2020;19(7):441–2.
Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, et al. Molecular engineering of functional siRNA agents. ACS Synth Biol. 2012;1(10):576–84.
Chen W, Zhou Y, Shi J, Xu S, Chen B, Sun H, et al. Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs. Appl Microbiol Biotechnol. 2019;103(5):2059–69.
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Iteration 3
Choosing the Delivery Platform
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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).
References
Sercombe L, Veerati T, Moheimani F, Wu SY, Sood AK, Hua S. Advances and Challenges of Liposome Assisted Drug Delivery. Front Pharmacol. 2015;6:286.
Allen TM, Cullis PR. Liposomal drug delivery systems: From concept to clinical applications. Adv Drug Deliv Rev. 2013;65(1):36–48.
Pattni BS, Chupin VV, Torchilin VP. New Developments in Liposomal Drug Delivery. Chem Rev. 2015;115(19):10938–66.
Hua S. Advances in nanoparticulate drug delivery approaches for respiratory disorders. Expert Opin Drug Deliv. 2014;11(6):845–56.
Kulkarni JA, Witzigmann D, Chen S, Cullis PR, van der Meel R. Lipid Nanoparticle Technology for Clinical Translation of siRNA Therapeutics. Acc Chem Res. 2019;52(9):2435–44.
Bergenfelz C, Do P, Larsson L, Ivarsson H, Malmborn K, Håkansson AP. Corynebacteria from the respiratory microbiota modulate inflammatory responses and are associated with a reduced pneumococcal burden in the lungs. Front Cell Infect Microbiol. 2025;14:1530178. doi:10.3389/fcimb.2024.1530178.
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Iteration 4
Selecting the Final Chassis
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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: firstly, it acts as a chassis for CO-BERA delivery. Secondly, the L.plantarum can be a therapeutic agent due to its anti-inflammatory function. This made it the ideal choice for our final system.
References
Becker J, Wittmann C. Systems and synthetic metabolic engineering for amino acid production – the heartbeat of industrial strain development. Curr Opin Biotechnol. 2012;23(5):718-26.
Bergenfelz C, Do P, Larsson L, Ivarsson H, Malmborn K, Håkansson AP. Corynebacteria from the respiratory microbiota modulate inflammatory responses and associate with a reduced pneumococcal burden in the lungs. Front Cell Infect Microbiol. 2025;14:1530178. doi:10.3389/fcimb.2024.1530178.
Lee J, Kim Y, Yun HS, Kim JG, Oh S, Kim SH. Genetic and proteomic analysis of anti-inflammatory functions of Lactobacillus plantarum 88 in IL-10-deficient mice. J Appl Microbiol. 2016;120(6):1700-12.
Zhou Y, Cui Y, Qu X. Lactobacillus plantarum inhibits intestinal inflammation and regulates gut microbiota in DSS-induced colitis mice. Exp Ther Med. 2019;18(3):2243-51.
Plaza-Díaz J, Ruiz-Ojeda FJ, Gil-Campos M, Gil A. Mechanisms of action of probiotics. Adv Nutr. 2019;10(suppl_1):S49–S66.
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Optimizing Reliability
As the project progressed, we found that therapeutic RNA expression was insufficient to ensure reliable performance. Further optimization of accuracy, efficiency, and safety was required for our system. The gene expression should become more specific, the RNA cargo delivered efficiently inside host cells, and a safety system incorporated to prevent possible unintended risks.
Through design iterations, we added further layers of regulation for conditional expression, investigated means for enhancing intracellular delivery and functionality, and incorporated biocontainment through engineering to enhance safety. These improvements transformed the original idea into a much more robust, targeted, and responsible therapeutic platform.
Iteration 5
Precision Expression through Oxidative & pH Control
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Design
From initial design of expression of our approach we decided to placed CO-BERA under the control of the inducible promoter, which is activated by inflamed lung. We expected this design to provide disease-specific activation in asthma.
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, since elevated oxidative stress (H₂O₂) changes in inflamed airways. The KatA promoter, which is repressed under normal conditions by PerR. However, it is induced upon the presence of reactive oxygen species which 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:
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.
References
Ceroni F, Algar R, Stan GB, Ellis T. Quantifying cellular capacity identifies gene expression designs with reduced burden. Nat Methods. 2015;12(5):415-8. doi:10.1038/nmeth.3339. PMID: 25849635; PMCID: PMC4207942.
Lee JW, Helmann JD. Functional specialization within the Fur family of metalloregulators. Biometals. 2007;20(3-4):485-99.
Faulkner MJ, Helmann JD. Peroxide stress elicits adaptive changes in bacterial metal ion homeostasis. Antioxid Redox Signal. 2011;15(1):175-89.
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About our loading
While developing our RNA therapeutic system, we recognized that loading efficiency into bacterial membrane vesicles (MVs) is a major challenge. Although Lactobacillus plantarum can naturally release vesicles that carry biomolecules, only a small fraction of the engineered RNA (CO-BERA) was successfully packaged. This limitation reduces the therapeutic payload and weakens clinical applicability. Therefore enhancing the proportion of RNA molecules loaded into vesicles became a key design goal. To address this, we explored biological strategies that could actively guide RNA toward vesicle formation sites, rather than relying on passive or random encapsulation.
Iteration 6
Increasing RNA Loading Efficiency
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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
Initial analysis suggested and after deep search we found that however, CSP could indeed bind RNA, but its role in vesicle loading was uncertain. As it is cytoblasmic protein which is not ensure the loading efficacy.
Learn
We decided to search more about another RNA binding protein and integrated to membrane could serve as a localization factor and ensure packaging of RNA into vesicles.
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Iteration 7
Searching for a Membrane-Bound RNA Binding System
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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
We decided to design protein complex 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.
The RMSD plot of the Foldase PrsA complex shows a steady increase over the 5 ns simulation, indicating progressive structural deviation from the initial conformation. This reflect dynamic flexibility or instability under simulated conditions.
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.
this is 3D structure prediction of DUF4811-L7Ae
Re-Test
Computational modeling with SWISS-Model and CHARMM-GUI, followed by GROMACS molecular dynamics simulations, confirmed stable membrane orientation.
Figure: This density to RMSD graph shows the huge difference between the stability of two TMP linked to L7Ae (RBP)
Learn
This established DUF4811–L7Ae as a stable and reliable RNA loading system, supporting efficient localization of RNA to vesicles.
Iteration 8
Optimizing C/D Box Motif for CO-BERA Loading Efficiency
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Design
To enhance the loading efficiency of CO-BERA RNA into bacterial membrane vesicles (BMVs), we aimed to optimize the C/D Box motif configuration, which serves as the recognition site for the L7Ae RNA-binding protein [1]. Initially, we designed CO-BERA with two C/D Box motifs at the 3′ end, inspired by the iGEM 2024 HBMU-Taihe team, which reported that diagnostic RNAs with two C/D Boxes achieved superior vesicle loading [8].
Build
We designed our genetic circuit based on using two C/D Box motifs at the 3′ end of CO-BERA to facilitate L7Ae binding and BMV packaging [2]. The CO-BERA construct was incorporated into an L. plantarum expression vector, compatible with our DUF4811-L7Ae fusion protein for RNA localization to BMVs [3].
Test
After conducting molecular dynamics simulations using CHARMM-GUI and OpenMM, we found that a single C/D Box motif exhibited a higher docking score and lower ensemble diversity compared to the two C/D Box configurations [4, 5]. The simulations suggested that two motifs may introduce RNA folding constraints, reducing loading efficiency into BMVs.
Learn
The simulation results indicated that a single C/D Box motif may outperform the two C/D Box design for CO-BERA loading into BMVs due to enhanced L7Ae accessibility. To validate this finding empirically, we designed and ordered DNA fragments encoding CO-BERA constructs with varying numbers of C/D Box motifs (one, two, and three) from Twist Bioscience. These fragments include C/D Box sequences at the 3′ end of CO-BERA and are designed for Golden Gate cloning into our L. plantarum expression vector [6]. Our lab experiments will involve transforming these constructs into Lactobacillus plantarum, inducing BMV production, and quantifying RNA loading efficiency via qRT-PCR to confirm the optimal C/D Box configuration [7].
References
Fabbiano F, Notarangelo M, Corsi J, et al. RNA packaging into extracellular vesicles: An orchestra of RNA-binding proteins? J Extracell Vesicles. 2020;9(1):12043. doi:10.1002/jev2.12043.
Rozhdestvensky TS, Tang TH, Tchirkova IV, Brosius J, Bachellerie JP, Hüttenhofer A. Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea. Nucleic Acids Res. 2003;31(3):869–877. doi:10.1093/nar/gkg175.
Velez M, Arluison V. Does the Hfq Protein Contribute to RNA Cargo Translocation into Bacterial Outer Membrane Vesicles? Pathogens. 2025;14(4):399. doi:10.3390/pathogens14040399.
Jo S, Kim T, Iyer VG, Im W. CHARMM-GUI: A web-based graphical user interface for CHARMM. J Comput Chem. 2008;29(11):1859–1865. doi:10.1002/jcc.20945.
Eastman P, Swails J, Chodera JD, et al. OpenMM 7: Rapid development of high performance algorithms for molecular dynamics. PLoS Comput Biol. 2017;13(7):e1005659. doi:10.1371/journal.pcbi.1005659.
Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One. 2008;3(11):e3647. doi:10.1371/journal.pone.0003647.
Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–622. doi:10.1373/clinchem.2008.112797.
After establishing the optimized conditional expression and packaging of our CO-BERA therapeutic system within engineered Lactobacillus plantarum, we aimed to ensure that the therapeutic RNA could reach its functional site inside host cells. While previous iterations optimized sensing, regulation, and delivery to airway epithelial cells, intracellular release remained a challenge. Once bacterial membrane vesicles deliver CO-BERA into the host cell, most RNA cargos become trapped within endosomes, which reduces their silencing efficiency. Therefore, the next cycle was to enhance endosomal escape to maximize cytoplasmic delivery of CO-BERA and ensure its full therapeutic potential.
Iteration 9
Enhancing endosomal escape with Mutated LLO
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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 < 1% of RNA cargos naturally escape endosomes which severely limits 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)..
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.
Wild LLO : Degradation of CO-BERA at lysosomal or lat endosomal pH as wild LLO become active at low pH.
L461T LLO : early activation of Mutated LLO so preserve CO-BERA from degradation.
References
Roth AJ, Zoller N, Bakowska-Polak A, Behrens HM, Klussmann S. Delivery of RNA therapeutics: the great endosomal escape! RNA Biol. 2023;20(1):214–227. PMID: 36372998; PMCID: PMC9595607.
Resnik N, Tratnjek L, Erdani-Kreft M, Kisovec M, et al. Engineering a pH-responsive pore-forming protein. Sci Rep. 2017;7:42231. doi:10.1038/srep42231.
Carriero M, Lanni F, De Simone M, Falcigno L, Matarazzo MR, Pane F, et al. Carboxyl-terminal residues N478 and V479 required for hemolytic activity of listeriolysin O. Front Immunol. 2017;8:1439. doi:10.3389/fimmu.2017.01439.
Geoffroy MC, Gaillard JL, Alouf JE, Berche P. Purification, characterization, and toxicity of the sulfhydryl-activated hemolysin listeriolysin O from Listeria monocytogenes. Infect Immun. 1987;55(7):1641-6. PMID: 3110068; PMCID: PMC260565.
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Iteration 10
Advancing Biosafety with a Toxin–Antitoxin (TA) System
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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 toxin and 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 exposed to (phosphate as in blood stream or Low temperature as in external environment), the antitoxin (PemI) which is less stable will be degraded, while the stable toxin (PemK) remains active—leading to self-elimination of the escaped bacteria (2,3).
Test
After our model validation and 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.
Figure:
This figure shows PemI/K kill switch component changes over time equal to 10 hours and the deactivation of the
circuit at time (5h) that leads to decreased complex and antitoxin concentration and toxin accumulation.
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).
The illustrations show the killing of our bacteria in unintended environment
References
Plaza-Díaz J, Ruiz-Ojeda FJ, Gil-Campos M, Gil A. Mechanisms of action of probiotics. Adv Nutr. 2019;10(suppl_1):S49–66. doi:10.1093/advances/nmy063.
Harms A, Brodersen DE, Mitarai N, Gerdes K. Toxins, targets, and triggers: an overview of toxin–antitoxin biology. Mol Cell. 2018;70(5):768–784. doi:10.1016/j.molcel.2018.01.003.
Kato F, Yoshizawa S, Yamaguchi Y. New insights into toxin–antitoxin systems in bacteria: biochemistry and physiology. Front Microbiol. 2023;14:1178321. doi:10.3389/fmicb.2023.1178321
Chan CTY, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol. 2016;12(2):82–86. doi:10.1038/nchembio.1979.
Stirling F, Bitzan L, O'Keefe S, Redfield E, Oliver JW, Way J, Silver PA. Rational design of evolutionarily stable microbial kill switches. Mol Cell. 2017;68(4):686–697.e3. doi:10.1016/j.molcel.2017.10.033.
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Optimizing Bacterial Delivery
Our goal is to deliver our engineered Lactobacillus plantarum effectively into the lungs, and this forms the basis of therapeutic treatment. The method of delivery should preserve bacterial viability and stability, provide efficient deposition in the respiratory tract, and be a viable option when considered from a practical point of view for patients. To address these requirements, we searched about different pulmonary delivery strategies and refined our approach through iterative design and evaluation. This process leads us toward a delivery system that balances effectiveness, safety, and scalability for real clinical application.
Iteration 11
Delivery Method Selection
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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.
Graph showing how particle size and geometric standard deviation (GSD) affect lung deposition efficiency. The current design (~3 μm MMAD, GSD = 2.0) achieves ~25% deposition, supporting its suitability for alveolar targeting in dry powder inhaler formulations.
Learn
Following discussions with Dr/ Suzan, we transitioned to Dry Powder Inhalers (DPI) as the delivery platform. DPIs provided several advantages (5):
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.
References
Laube BL. The expanding role of aerosols in systemic drug delivery, gene therapy, and vaccination. Respir Care. 2005 Sep;50(9):1161-76. PMID: 16122400.
Darquenne C. Deposition mechanisms. J Aerosol Med Pulm Drug Deliv. 2020 Apr;33(2):61-5. doi:10.1089/jamp.2019.1558.
Carvalho TC, Peters JI, Williams RO 3rd. Influence of particle size on regional lung deposition – what evidence is there? Int J Pharm. 2011 Sep 30;406(1-2):1-10. doi:10.1016/j.ijpharm.2010.12.040.
Loretz B, Schäfer J, Vogt L, Winter G, Lehr CM. Pulmonary delivery of therapeutic proteins. J Aerosol Med Pulm Drug Deliv. 2011 Jun;24(3):127-35. doi:10.1089/jamp.2010.0859.
Vandenheuvel D, Meeus J, Lavigne R, Van den Mooter G. Instability of bacteriophages in spray-dried trehalose powders is influenced by the stress imposed by spray-drying. Int J Pharm. 2013 Jun 15;450(1-2):128-36. doi:10.1016/j.ijpharm.2013.04.020.
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Iteration 12
preparation of the powder
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Design
To ensure effective delivery of our engineered Lactobacillus plantarum via Dry Powder Inhalers (DPIs), we aimed to develop a scalable and stable formulation that preserves bacterial viability and ensures efficient lung deposition. Initially, we selected spray-drying as the formulation method due to its fast, continuous, and widely used industrial process for producing dry formulations, making it attractive for scalable therapeutic production [1]. We designed the process to generate fine dry powder suitable for inhalation, targeting a mass median aerodynamic diameter (MMAD) of 1–5 μm for optimal alveolar deposition [2].
Build
We explored spray-drying conditions commonly used for probiotics, where heated air rapidly evaporates the solvent to produce a fine dry powder compatible with DPI devices [3]. We incorporated protective excipients like trehalose to stabilize L. plantarum during drying, aiming to minimize thermal stress and maintain cell integrity [4]. The process was designed to optimize parameters such as inlet temperature (120–150°C) and feed rate based on literature recommendations for probiotic formulations [5].
Test
Further research and literature-based modeling revealed significant drawbacks of spray-drying for our application:
High temperature exposure (120–150°C) reduced probiotic viability, with survival rates dropping to 59.5% due to thermal stress [6].
Rapid dehydration damaged bacterial membranes, leading to compromised cell integrity [7].
Poor long-term survival rates limited clinical feasibility, as powders showed reduced stability during storage [8].
As our model validated these findings, where spray-dried L. plantarum exhibited a viability loss of ~40% (measured via colony-forming unit (CFU) counts) compared to controls. In contrast, freeze-drying achieved a survival rate of 72.7%, a 13.2% improvement over spray-drying, as confirmed by in vitro assays. These results aligned with literature highlighting freeze-drying's superiority for probiotic preservation [9].
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
Based on our modeling results, preliminary experimental data, and guidance from Dr. Ahmed Abdelsabour, we transitioned to freeze-drying (lyophilization) as the preferred method for DPI formulation. Freeze-drying offers:
Superior preservation of bacterial viability compared to spray-drying, minimizing thermal and osmotic stress [6].
Lower stress on bacterial membranes during processing, maintaining cell integrity [9].
Compatibility with protective excipients (e.g., trehalose, mannitol) that enhance powder stability and aerosol dispersion [10].
This choice ensures that Lactobacillus plantarum remains viable throughout production, storage, and delivery, aligning with patient-friendly DPI systems used in asthma management.
References
Sosnik A, Seremeta KP. Advantages and challenges of the spray-drying technology for the production of pure drug particles and drug-loaded polymeric carriers. Adv Colloid Interface Sci. 2015;223:40–54. doi:10.1016/j.cis.2015.05.003.
Labiris NR, Dolovich MB. Pulmonary drug delivery. Part I: Physiological factors affecting therapeutic effectiveness of aerosolized medications. Br J Clin Pharmacol. 2003;56(6):588–599. doi:10.1046/j.1365-2125.2003.01892.x.
Broeckx G, Vandenheuvel D, Claes IJ, Lebeer S, Kiekens F. Drying techniques of probiotic bacteria as an important step towards the development of novel pharmabiotics. Int J Pharm. 2016;505(1-2):303–318. doi:10.1016/j.ijpharm.2016.04.002.
Morgan CA, Herman N, White PA, Vesey G. Preservation of micro-organisms by drying; a review. J Microbiol Methods. 2006;66(2):183–193. doi:10.1016/j.mimet.2006.02.017.
Huang S, Vigneswari S, Ng IS, Chai C, Chen PT. Spray-drying of probiotics and its application in food and healthcare industries. Crit Rev Food Sci Nutr. 2022;62(23):6325–6342. doi:10.1080/10408398.2021.1906624.
Santivarangkna C, Kulozik U, Foerst P. Inactivation mechanisms of lactic acid starter cultures preserved by drying processes. J Appl Microbiol. 2008;105(1):1–13. doi:10.1111/j.1365-2672.2007.03670.x.
Peighambardoust SH, Golshan Tafti A, Hesari J. Application of spray drying for preservation of lactic acid bacteria. Food Rev Int. 2011;27(1):80–102. doi:10.1080/87559129.2010.496197.
Carvalho AS, Silva J, Ho P, Teixeira P, Malcata FX, Gibbs P. Effects of various sugars added to growth and drying media upon thermotolerance and survival throughout storage of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus. Biotechnol Prog. 2004;20(1):248–254. doi:10.1021/bp034165n.
Leslie SB, Israeli E, Lighthart B, Crowe JH, Crowe LM. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl Environ Microbiol. 1995;61(10):3592–3597. doi:10.1128/aem.61.10.3592-3597.1995.
Maa YF, Prestrelski SJ. Biopharmaceutical powders: Particle formation and formulation considerations for inhalation delivery. Curr Pharm Biotechnol. 2000;1(3):283–302. doi:10.2174/1389201003378897.
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Iteration 13
Fragment Preparation & Validation
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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.
Figure: electrophoresis showing the size of fragments 1,2 and 3.
