Before showing our art of choosing a perfect design with its safety considerations, we need to let you know about this Lac operon, which is natively located in the bacteria. The lac operon consists of a promoter (P) and operator (O) region followed by three structural genes lacZ, lacY, and lacA downstream. A regulatory gene lacI (I) preceding the lac operon is responsible for producing a repressor (R) protein.
The structure of lac operon
The lac promoter is located at the 5′ end of lacZ and directs transcription of all three genes as a single mRNA. This mRNA is translated to give three protein products (shown in the table below):
| Structural gene | Enzyme | Function |
|---|---|---|
| lacZ | β-galactosidase (B) | It transforms lactose into allolactose and also catalyzes the conversion of lactose to glucose and galactose. |
| lacY | permease (P) | Membrane channel protein required to uptake lactose from the environment |
| lacA | thiogalactoside transacetylase | It rids the cell of toxic thiogalactosides that also get transported by lacY. |
Normally, lacI, which is located prior to the lac, produces a repressor (LacR) protein that inhibits the Lac operon by binding to its operator, stopping the expression of lac operon proteins, and this scenario occurs in the case of abscence of lactose in the bacteria, and if there are some lactose molecules in our bacteria these molecules will transform into allolactose and bind to the LacR, preventing its function, and derepressing the lac operon proteins expression.
LacR binds to Lac operon operator, inhibiting its proteins expression and metabolism
LacR is inhibited by the allolactose, preventing it from repressing the operon
Skim milk and trehalose are cryoprotectants, frequently used in freeze drying while preparing capsules for dry powder inhalers (DPIs) due to their stability, biocompatibility, and ability to enhance powder dispersion [1]. In our project, we uses L. plantarum, which is lactic acid bacteria can metabolize the lactose into lactic acid.
This metabolism cause lactic acid production and degradation of our particles' matrix structure [5], decreasing the particles' half-life in the lung, reducing both patient compliance and therapeutic efficacy by preventing particles' deposition while maintaining an acidic pH in the lung, causing exacerbation of airway acidity, and irritation.
Skim milk and trehalose digestion mechanism
Trehalose, a non-reducing disaccharide, can be metabolized via trehalase, producing glucose that may aid in the production of lactic acid [4]. A prior study showed that when L. plantarum LIP-1 was cultured to the stationary phase with trehalose supplementation, the environmental pH was significantly lower than in the control group over 2–6 hours, causing acid stress [4]. · The skim milk is rich with lactose that will be metabolized into lactic acid.
To maintain matrix integrity and prevent or literally delay skim milk consumption, we used the first two components of the CO-BERA circuit with ther secondary function, which are the p170_CP25 hybrid promoter, a pH-responsive sensor activated by acidic conditions in asthmatic airways (< pH 6.9), and the second part is LacR that prevents the skim milk matrix's metabolism and its consequences, minimizing lactic acid production and preserving the matrix's structural integrity for more time [5]. This extends the particle's half-life at the storage and in the lung, improves patient compliance, enhances therapeutic efficacy by ensuring reliable probiotic deposition, and maintains a neutral pH in the lung, preventing exacerbation of airway acidity, reducing irritation, and supporting L. plantarum's anti-inflammatory effects [6].
This mechanism prevents early particle digestion during capsule storage or during aerosolization [8], ensuring effective delivery and preserving bacterial survival until deposition in the lung. By addressing the variability in disease severity and flare-ups, this mechanism allows L. plantarum to accurately control the expression of both LacR and CO-BERA, offering a flexible therapy responsive to the changeable pH environment of asthmatic airways. Unlike constitutive promoters that activate uniformly, this promoter ensures targeted, context-specific activation. It improves targeting of TSLP-driven inflammation, improving efficacy for patients with uncontrolled asthma by reducing immunogenic hazards, such as Th2-driven reactions or airway irritation, which are serious issues in patients suffering from asthma with compromised immune systems [9].
Artificially changes can be easily induced in vitro (e.g., in labs) by adding inducers like isopropyl β-D-1-thiogalactopyranoside (IPTG), with skim milk to L. plantarum that facilitates safety assessments under artificially simulated real condition when the bacteria metabolize the lactose of skim milk into lactic acid, as IPTG binds the LacR and let the lactose be metabolized, that saves time to us and enables optimization of the genetic circuit before testing in vivo, giving our team confidence in our system's reliability and safety profile [10].
We observed that it will be the most similar inducer as it acts with the same mechanism, giving the bacteria the chance to degrade its matrix. Unlike lactose, IPTG is not part of any metabolic pathways and will not be broken down or metabolized by the cell. This ensures that the concentration of IPTG we add remains constant and will not affect the results, making it the most useful inducer of the lac operon [30].
IPTG mimics the allolactose and simulate the lactose digestion mechanism in the lung
We use isopropyl β-D-1-thiogalactopyranoside (IPTG) as a synthetic analog of allolactose that binds to LacR with high specificity (Kd ≈ 10⁻⁶ M). · Upon IPTG binding, the repressor undergoes conformational changes that reduce its DNA-binding affinity by approximately 1000-fold (Kd = 10⁻¹⁰ M) [26], effectively derepressing the lac operon and triggering transcription of lacZYA genes encoding β-galactosidase, permease, and transacetylase. · Our team found that the best bacterial growth and lactic acid production were achieved at pH = 6.5 [27]. · We determined that IPTG is effective in the concentration range of 100 μmol/L to 3.0 mmol/L [28]. Following GoldBio's protocol, we recommend using 1mM of IPTG in 1 ml of LB medium to make a final concentration of 0.5mM in the medium with bacterial culture [29].
Unlike natural RNA molecules, CO-BERA lacks a "parent organism" or inherent biological function in nature [12]. The primary risk is the off-target effects, as the siRNA may bind to and silence unintended human mRNAs rather than the TSLP. This could lead to cellular toxicity, disrupted cellular processes, or other unpredictable physiological complications [13].
To ensure both safety and specificity in our project, we have created a multi-layered risk management plan that includes the CO-BERA load and the delivery method. And our great multi-system includes:
Using siDirect 2.0 software, we designed CO-BERA with the least off-target effects, selecting sequences with high specificity for TSLP mRNA and the least off-target probability for lung tissue mRNAs, as it may target other mRNAs outside the lung [14, 15]. This reduces any unintended cellular effects and ensures selective TSLP silencing.
siRNA computational screening process
Our team designed an impressive RNA delivery system that uses a two-part loading mechanism combining RNA-binding proteins (RBPs) with engineered RNA sequences, which is CO-BERA. This approach ensures that only our therapeutic RNA containing specific sequences (such as the K-turn motif recognized by L7Ae) can be packaged into bacterial membrane vesicles.
We believe that this selectivity significantly reduces unintended effects and prevents the accidental inclusion of cellular RNA that prevents unwanted biological responses, addressing key safety concerns in RNA therapeutics [31, 32].
Through the binding interaction between L7Ae and the C/D Box motif, we observed that our CO-BERA achieves improved stability during transporting and storage. This stability improvement not only maintains therapeutic effectiveness but also prevents the release of broken RNA fragments that could trigger immune responses or cellular damage.
We found this enhanced stability is particularly beneficial for dry powder inhaler formulations containing trehalose and skim milk matrices [1]
Our development process incorporated thorough computational screening using CHARMM-GUI for system building and GROMACS for molecular dynamics analysis. We systematically evaluated multiple membrane protein candidates (Foldase PrsA and DUF4811), ultimately selecting DUF4811-L7Ae based on superior membrane stability.
This computational approach helped us reduce risks associated with protein separation, membrane disruption, or structural instability during vesicle formation and delivery [32].
We chose bacterial membrane vesicles because they represent naturally occurring cellular structures, providing inherent compatibility advantages over synthetic delivery vehicles. Our usage of well-characterized archaeal proteins like L7Ae offers predictable binding behavior and reduces the risk of unexpected protein interactions or immune recognition [32, 33].
These integrated safety measures create a comprehensive risk management framework that our team developed to address potential hazards at multiple levels, from molecular design through environmental deployment, ensuring both therapeutic effectiveness and patient safety in using CO-BERA.
The enhanced loading efficiency achieved through our engineered system allows for lower therapeutic doses while maintaining effectiveness. We anticipate this dose reduction will directly translate to decreased potential for adverse effects and improved patient safety profiles, which is particularly important in asthma where local tissue sensitivity is a major concern [5, 6].
LLO is a virulence factor in its native state, it creates holes in the phagosomal membrane, allowing bacteria to pass through [13]. This makes it simple for L. monocytogenes to enter the cytoplasm of the host cell and start infection. LLO's pore-forming activity is naturally maximized at the acidic pH of the phagosome (pH 5.5–6.0) but quickly deactivated at neutral cytoplasmic pH [17, 18]. Serious listeriosis symptoms, including septicemia, meningitis, and neonatal infections, are brought by the cytotoxicity of LLO in its original wild form [19].
By creating the LLO-L461T mutant and putting strict regulatory measures in place to guarantee safety and specificity, we were able to use LLO's membrane-permeabilizing capabilities for therapeutic purposes while reducing its toxicity, and making it acts at neutral pH (pH 6.5) rather than acidic pH, as the CO-BERA is sensitive to acidity. We reduced its over-expression and off-targeting by expressing LLO-L461T under the regulation of pKatA, which senses high concentration of H2O2 (< 10 µM) [20].
Leucine is swapped out for threonine at position 461 in the LLO-L461T mutant, which is a single amino acid change [21]. While maintaining LLO's capacity to temporarily permeabilize membranes, this mutation dramatically diminishes its hemolytic properties and effects [21]. The L461T mutation reduces pathogenicity by about 100 times when compared to wild-type LLO, while maintaining the creation of large and stable pores [22]. LLO-L461T is perfect for applications like targeted drug delivery or immunological regulation because, despite its attenuation, it still has enough pore-forming activity to permit controlled endosomal escape [23].
To enhance safety and specificity, we regulated hly gene encoding LLO-L461T by a responsive promoter to environmental cue in the therapeutic context [7]. We decided to use the pKatA promoter to make the LLO-L461T be only formed in inflammatory microenvironments with high H₂O₂ levels (10–100 µM) as the hydrogen peroxide (H₂O₂) level is elevated in inflammed tissues, such as asthmatic airways [7, 24]. The mechanism stays repressed in non-target settings (low ROS), avoiding over-expression and off-target effects and unintended disruption of membranes in healthy tissues [25].
The LLO-L461T system is designed for applications like targeted drug delivery and immune modulation in asthma therapy [23]. LLO-L461T facilitates the delivery of CO-BERA directly into the cytoplasm by enabling controlled endosomal escape, enhancing efficacy, minimizing off-targeting, and preventing unintended actions [23]. Both the reduced pathogenicity and conditional expression mitigate some risks that are associated with the native wild-type LLO, such as excessive cell lysis or systemic toxicity [22].
LLO-L461T Expression Cascade
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