Our Inspiration story

During our brainstorming sessions at AFCM headquarters, team members were tossing ideas about what topic we would adopt this year.

Overview

Asthma is marked by a chronic inflammatory condition of the airways, which is associated with airflow obstruction that is reversible or partially reversible. It is also manifested with bronchial hyperresponsiveness and mucus hypersecretion. Clinically, the symptoms may vary from episodic cough and wheezing to a severe life-threatening attack (1,2,3). It is interrupted by acute attacks triggered by environmental stimuli. Despite asthma currently having no cure, controlling asthma symptoms is the main aim of the current treatment.

FIG (1): Showing symptoms of asthmatic patients.

Epidemiology

Asthma is characterized by unpredictable and debilitating flare-ups, affecting about 300 million people globally. This illness may begin in childhood and also in adults (4). The time of starting treatment is often delayed due to misdiagnosis; the delay increases morbidity, mortality levels, and quality of life. Poverty and the absence of medical insurance are also determinants that influence the number of asthmatics and caregivers. Despite all attempts to the contrary, we are still without a definitive answer as to what causes asthma, although it is broadly agreed upon that asthma manifests after a complex interaction between genetic and environmental exposures.

FIG (2): Showing asthma prevalence and mortality rates.

Pathogenesis

Asthma pathogenesis is based on the integration of genetic and environmental factors, with the interference of poverty and a lack of health insurance (5). While the exact cause of asthma development remains unclear. The pathogenic process in asthma constitutes a biphasic inflammatory response:

FIG (3): Showing the precipitating factors of asthma.

Ealry Phase

FIG (4): Showing the role of TSLP in asthma pathogenesis.

This phase begins shortly after trigger exposure, which activates airway epithelial cells to release alarmins: IL-25, IL-33 and Thymic Stromal Lymphopoietin (TSLP) (6). Significantly, TSLP stimulates dendritic cells (7,8), promoting:

• Th2 lymphocytes by differentiation of naïve T cells.
• plasma cells by B-cell maturation

Subsequently, Th2 cells and plasma cells secrete IL-4, IL-5, and IL-13 (9).

FIG (5): Showing the effect of inflammatory cytokines on IgE.

IgE binds to FcεRI receptors on mast cells, triggering degradation and release of pro-inflammatory mediators. This leads to smooth muscle contraction and airway narrowing (6). IgE also participates in the recruitment of the late-phase reactant (10).

Late Phase

This phase occurs hours post trigger, marked by recruitment of eosinophils, basophils, neutrophils, and memory T cells to airway tissues (10). This influx precipitates sustained inflammation that leads to:

• Bronchoconstriction
• Airway edema
• Mucus hypersecretion
• Structural remodeling

FIG (6): Showing the airways structural remodeling.

These changes contribute to persistent airflow limitation, which may become irreversible over time.

FIG (7): Showing the airway narrowing of the asthmatic patient.

Risk factors

There are many factors for an increased risk of developing asthma. In addition, all these factors might lead to a greater severity of symptoms and increase the number of emergency and hospital admissions for asthmatics. However, not all of these factors have the same effect on each patient.

FIG (8): Showing the risk factors that may trigger flare-ups of asthma.

Classification of Asthma

Asthma can be classified based on the underlying mechanism of disease progression, disease severity and the nature of the trigger factor involved.

FIG (9): Showing classification of asthma according to triggers, severity and mechanism of disease.

FIG (10): Showing the guidelines used for asthma classification.

According to symptoms such as frequency of symptoms, limitations in activity, and nighttime awakening, and the results of respiratory function tests, severity of asthma is classified into four main groups: intermittent, mild persistent, moderate persistent, and severe persistent.

The classification of an asthmatic patient should be established before selecting the treatment plan to maximize the benefit of this approach, since it becomes more difficult to subgroup after treatment has begun. The severity of asthma must also be assessed periodically, by monitoring symptoms and limitations of activity of the patient, to evaluate their risk for a future attack.

FIG (11): Showing the GINA guideline for the classification of asthma.

Asthma diagnosis

Asthma is suspected with episodic symptoms of airflow obstruction, which can be partially reversible or permanent. A comprehensive approach for asthma diagnosis that includes personal and medical history, a physical examination and lung function tests. Additional diagnostic tools such as allergy and blood tests. These diagnostic methods are generally accessible and represent the ideal approach. Furthermore, this approach can be performed easily in a clinical setting.

FIG (12): Showing the comprehensive approach for asthma diagnosis.

Diagnosis of asthma is confirmed by lung function tests, which are designed to assess the patient’s ability to inhale and exhale air. Furthermore, they assess breathing effort and evaluate the overall lung capacity. Ideally, lung function tests are measured both before and after inhalation of a bronchodilator to assess improvement in airflow. A significant improvement in post-bronchodilator measurement usually suggests the presence of asthma.

History of asthma treatment

FIG (13): Showing the asthma treatment throughout ages

Current treatments

Traditional Treatment

Asthma prevalence continues to rise every year. Despite the numerous established asthma medications, they focus on managing asthma symptoms rather than addressing the underlying pathology of the illness. Severe asthma is characterized by frequent attacks and concomitant drug intake. Consequently, it is hard for asthmatic patients to adhere well to their management. Consequently, long-term application of such established medicines may expose the patients to numerous undesirable outcomes.

Relievers (Quick-acting):

• Short-acting β2 agonists (SABA) like salbutamol.
• Provide rapid relief of acute symptoms.

Controllers (Long-term):

• Inhaled corticosteroids (ICS) are the mainstay for reducing airway inflammation.
• Long-acting β2 agonists (LABA) – often combined with ICS.
• Leukotriene receptor antagonists (LTRAs).
• Theophylline (less commonly used due to side effects).

Limitations

• Side effects (oral thrush, tremors, systemic effects at high doses).
• Some patients remain uncontrolled despite optimal therapy.
• Do not fully address disease heterogeneity.

Biological therapy

Biologic therapies are a recent technique applied in treating patients with severe or uncontrolled asthma poorly responsive to conventional therapy. Compared with usual drugs that act broadly, the biological molecules exclusively act against one or multiple site specific immune mediators causing asthma pathogenesis. For example, omalizumab neutralizes IgE (24,25), while tezepelumab blocks the epithelial cytokine TSLP (26,27). These drugs have shown the ability to prevent rather than merely control exacerbations and thus permit better control of symptoms (28), while withdrawal of these drugs usually causes the asthma symptoms to recur, ensuring that biologics administration must be at least once a week to prevent asthma recurrence. Apart from the positives of the agents, there are some drawbacks, including excessive cost, poor availability, systemic application, invasive administration requiring injections, and a slow onset of activity, making them not ideal for offering quick-acting effects. Moreover, injections are coupled with hazards necessitating monitoring of the patient for the development of adverse effects such as injection site reactions, allergic reactions, recurrent infection, headaches, fatigue, and musculoskeletal pain (29).

FIG (14): Showing the current medications for asthma with their adverse effects.

It is evident from the above that the present therapies are usually coupled with significant side effects and the pathogenesis of asthma continues to affect the quality of patients’ lives. To counter these issues, we must come up with an upgraded therapeutic strategy that not only manages symptoms efficiently but also addresses the fundamentals of the illness while cutting down on complications. In response to this need, we developed PRESS. PRESS is a novel therapeutic technique that was born out of overcoming the limitations of existing therapies and provides a safer, more sustainable solution for asthmatic patients.

FIG (15): Showing the side effects that affect the asthmatic patient.

Project inspiration

Our approach is inspired by NJU-China 2021's therapeutic strategy for asthma. Their approach is based on knocking down both TSLP and GATA3 to inhibit type 2 inflammation. This knockdown of the two previous targets relies on interference (siRNA) technology. Significantly, the siRNA based genetic circuits are delivered to the lung epithelial cell via liposome mediated delivery, which is administered through nasal inhalation. Once inside the cells, these circuits express siRNA, which is then encapsulated in exosomes secreted by the epithelial cells. Subsequently, these exosomes are transported to the specific cells involved in the inflammatory process of asthma, leading to knockdown of the corresponding protein expression to achieve their inhibitory effects.

However, the NJU-China approach exhibits several drawbacks. Primarily, siRNA is an inherently unstable molecule, making it susceptible to degradation by cellular endosomes. Additionally, the continuous expression of siRNA is irrelevant to the episodic nature of asthma flare-ups. Furthermore, the lack of a specific technology for loading siRNA into exosomes can diminish the approach's efficiency, consequently increasing the required dosage. Collectively, these factors restrict the therapeutic potential of siRNA, resulting in restriction of the overall efficacy of the NJU-China approach. This underscores a clear need for strategic modifications to optimize the approach.

Our solution

Unlike existing biologics, which require frequent systemic administration and are costly, our team developed PRESS (Programmable Respiratory microbiome as an Endogenous Sustainable System for drug delivery). PRESS is a live therapeutic system designed to be an asthma therapy, which is characterized by a locally-activated, targeted and sustainable alternative to current asthma treatments.

FIG (16): Showing the abbreviation of PRESS.

Our approach is based upon two core principles:

1-Using Probiotics as a Therapeutic Platform:

We employ the probiotic bacterium Lactobacillus plantarum, a safe and natural bacterium that is linked to the gut–lung axis. It is not only a source of native anti-inflammatory protection but is itself a programmable vehicle for the delivery of designed genetic circuits.

2-Targeted Silencing of Key Inflammatory Signals:

We adopted the challenge of designing genetic circuits to sense a specific inflammatory biomarker under asthma inflammatory conditions. Subsequently, L. Plantarum would produce therapeutic RNAs that would silence Thymic Stromal Lymphopoietin (TSLP). TSLP is tightly implicated as an upstream cytokine in asthma initiation, airway remodeling and steroid resistance. By targeting TSLP, we can treat a range of asthma subtypes and achieve broader therapeutic coverage than traditional drugs.

With the dual-technologies, PRESS creates an asthma therapy base that is local, responsive, sustainable and safe.

Core component of our PRESS:

Conditioned expression

Lactobacillus plantarum is genetically modified for the conditioned expression of CO-BERA. The CO-BERA is an RNA scaffold designed to carry two distinct therapeutic siRNAs for the specific knockdown of TSLP protein through RNAi silencing. Our approach utilizes the pKat promoter for the conditioned expression of CO-BERA. The pKAT promoter is engineered to sense a specific inflammatory biomarker in the asthma environment called hydrogen peroxide (H2O2). Significantly, the level of H2O2 is markedly increased during asthma flare-ups. Consequently, CO-BERA expression is initiated under these conditions.

CO-BERA loading

Even though siRNAs’ action necessitates their delivery to the targeted tissues. Consequently, we have engineered the bacteria to express a Loading system that maximizes the loading of CO-BERA into the extracellular membrane vesicles (EMVs). These CO-BERA-loaded vesicles then bud from the bacterial cell membrane, loaded with CO-BERA, and subsequently enter the respiratory epithelial cells via endocytosis.

Endosomal escape

Furthermore, our genetic circuit is engineered to express a mutated version of the protein called Listeriolysin O (LLO). This version of LLO is crucial to prevent degradation of CO-BERA by the cellular endosomes by enhancing the early endosomal escape. Moreover, it is safer than the non-mutated version. Collectively, all of these factors increase the therapeutic efficiency of siRNA.

Multilayer safety system

Although our chassis bacterium (Lactobacillus plantarum) is generally regarded as safe and naturally associated with the gut–lung axis, there remains a potential risk of translocation into the bloodstream or unintended survival outside the host. To mitigate this, we integrated the toxin–antitoxin system as the core biosafety mechanism of our design. This safeguard prevents the persistence of engineered bacteria in unintended sites and lowers the risk of horizontal gene transfer (HGT), as mentioned in the Safety page.

Conclusion

Asthma is a condition that affects millions of individuals globally, as it is variable in severity. Current therapies are often multifaceted in their side effects and have variable therapeutic outcomes depending on asthma severity. To overcome these drawbacks, we engineered a novel therapeutic platform named PRESS. PRESS is based on the anti-inflammatory properties founded on respiratory microbiome and RNA interference technologies. Specifically, PRESS exerts effects via silencing of the TSLP mRNA either by inducing degradation or suppressing translation. Accordingly, PRESS is designed to alleviate asthma more effectively and with fewer adverse effects relative to current therapies. Significantly, PRESS offers some practical advantages, including dosing only once or twice a year instead of daily or frequent inhalation and injections. Additionally, PRESS has the potential to lower overall healthcare costs by preventing exacerbations and hospital stays. Moreover, our financial analysis has highlighted that compared to other biological therapies, PRESS has the potential to be less costly per year for a patient. (Refer to entrepreneurship financial analysis.)