Abstract

Osteoporosis is the most common bone disease in the world, affecting over 200 million people and leading to debilitating fractures that carry a significant economic burden. While current treatments can be effective, they are plagued by severe systemic side effects, such as osteonecrosis of the jaw and atypical fractures. And as well as high costs, which together result in poor patient adherence. This situation creates a critical need for a safer, localized, and sustainable therapeutic alternative.

Inspired by the emerging field of living biotherapeutics, we developed a novel approach to address these limitations by creating a localized, in-situ drug factory. To do this, we engineered the bacteria Escherichia coli W3110 to function as an extracellular melanin production system. Our strategy was to display the key enzyme tyrosinase tyr1 on the cell surface using an AIDA autotransporter to simplify product harvesting.

To assure the absolute biocontainment and environmental safety, we implemented a robust strategy. This strategy consists of a metabolic auxotrophy, achieved by knocking out the murI gene to create an obligate dependence on DL-glutamate, a metabolite absent in human tissues. This is combined with physical encapsulation within an alginate hydrogel matrix that is formulated with this essential nutrient. To rigorously validate this containment strategy and enable safe laboratory research, we also designed and built a novel complementation plasmid that can restore DL-glutamate synthesis.

For additional biosafety, we plan to integrate a CcdA/CcdB toxin-antitoxin system: the toxin (ccdB) is on the pAIDA-tyrosinase plasmid, while the antitoxin (ccdA) is integrated into the genome. This setup ensures that only plasmid-containing bacteria survive, as plasmid loss leads to toxin-induced cell death, minimizing risks of dissemination or gene transfer.

Together, this engineered system enables the efficient and continuous production of melanin, which is a potent antioxidant that neutralizes damaging reactive oxygen species at the fracture site. This process reduces inflammation and actively promotes bone regeneration. Meanwhile, our dual biocontainment system ensures the bacteria are unable to replicate or survive outside their protective hydrogel niche, undergoing osmotic lysis upon any potential escape.

Ultimately, our work establishes a foundational platform for a new class of living medicines aimed at bone regeneration. By enabling localized, sustained, and controllable drug delivery. This paves the way for intelligent and sustainable solutions to a wide array of diseases.

I/ Background: Osteoporosis, a Global Health Challenge

Osteoporosis is the most common bone disease worldwide, affecting over 200 million people. It is a pathology characterized by decreased bone mineral density and deterioration of bone microarchitecture, leading to increased fragility and a high risk of fractures [1].This skeletal weakening occurs due to an imbalance in the natural bone remodeling process, where bone resorption chronically outpaces bone formation.

This process is governed by two key cell types: osteoclasts, which break down old bone or damage bone. And osteoblasts are bone-building cells. They produce the collagen matrix and mineralize it with calcium and phosphate. Once trapped in the bone they’ve built, they become osteocytes.The osteocyte acts as a master regulator, signaling the need for bone repair or resorption by detected micro-damage in the bone. In osteoporosis, the balance is disrupted, bone degradation exceeds bone formation : the osteoclast activity overwhelms osteoblast activity. That leads to a loss of bone density and an increased risk of fractures.

It often progresses silently for decades, earning it the name "the silent disease," as individuals may not experience any symptoms until a fracture occurs.

Figure 1: Comparison between healthy bone (right) and osteoporotic bone (left) showing bone density loss and microarchitectural deterioration

The impact is colossal: 1 in 3 women and 1 in 5 men aged over 50 will experience osteoporotic fractures (International Osteoporosis Foundation).

According to the WHO, osteoporosis is "an established and well-defined disease that affects more than 75 million people in Europe, Japan, and the USA, and causes more than 2.3 million fractures annually in Europe and the USA alone" [2].

The consequences of these fractures extend far beyond immediate pain. They represent a major turning point in an individual's quality of life and independence. Hip fractures, in particular, are devastating; they often require surgical intervention and are associated with a significant loss of autonomy and a marked increase in mortality risk. Vertebral fractures can lead to chronic pain, loss of height, and a stooped posture, which can impair lung function and digestion [3].

With the global aging population, this is alarming. By 2050, the worldwide incidence of hip fractures due to osteoporosis is expected to increase by 310% in men and 240% in women compared to 1990 rates [4]. The number of hip fractures is projected to nearly double from 2018 to 2050. This projected surge will place an immense and unsustainable burden on healthcare systems and economies globally.

It is therefore crucial to develop sustainable solutions not only to treat but also to prevent this silent epidemic. This demands a paradigm shift towards a multi-faceted approach, encompassing public health education on nutrition and weight-bearing exercise, widespread access to bone density scanning for early detection, and the development of more effective and accessible treatments to mitigate the impending wave of fractures and preserve the mobility and dignity of our aging population.

II/ Current Treatments

Current pharmacological treatments for osteoporosis are primarily divided into two categories: antiresorptive agents, which slow the breakdown of bone, and anabolic agents, which actively stimulate new bone formation. While proven effective at reducing fracture risk, these therapies are often accompanied by complex side effect profiles, significant costs, and adherence challenges, making the benefit-to-risk balance a difficult clinical calculation.[3]

The most widely prescribed treatments are bisphosphonates (e.g., Alendronate, Risedronate). They function by binding to the bone surface and being internalized by osteoclasts, the cells responsible for bone resorption, inducing their apoptosis and thereby slowing bone loss. However, their use is associated with a range of adverse effects. These include severe esophageal irritation, debilitating bone and muscle pain, and two rare but serious conditions: osteonecrosis of the jaw (ONJ), a destruction of the jawbone that can occur after dental work (with an incidence of 0.01-0.1%), and atypical femoral fractures, which are unusual breaks in the thigh bone with a low but serious risk profile[5].

As an alternative, Selective Estrogen Receptor Modulators (SERMs) like Raloxifene offer a hormone-related approach. They mimic estrogen's beneficial effect on bone by inhibiting osteoclasts without acting on other tissues. The most significant drawback is a substantial increase in the risk of thromboembolic events, including deep vein thrombosis and pulmonary embolism, with studies showing a relative risk increase of nearly threefold [6]. Side effects like hot flashes also commonly occur.

For patients with severe osteoporosis, anabolic agents (e.g., Teriparatide, Romosozumab) represent a more potent option by directly stimulating bone-forming osteoblasts. Their side effects, however, are equally significant. These can include hypercalcemia (high blood calcium) and a theoretical risk of osteosarcoma observed in animal studies, which limits treatment duration to two years. Specifically, Romosozumab carries a black box warning for an increased risk of major adverse cardiovascular events.[3]

Compounding these clinical challenges is the staggering economic burden, which is a global concern. In the United States alone, the annual direct cost of osteoporotic fractures was estimated at $19 billion and is projected to rise to $25.3 billion by 2025 [7]. This national figure is a subset of a massive worldwide cost, which was estimated at approximately $130 billion for major osteoporotic fractures in the 27 EU countries plus the UK, Switzerland, and Norway in 2017, with Germany and Italy bearing the highest direct costs. These costs are exacerbated by the high price of innovative anabolic therapies, which can exceed $1,000 per month, creating significant barriers to patient access and adherence, ultimately undermining the effectiveness of treatment programs on a public health scale.[8]

In summary, while current treatments provide a necessary defense against fractures, their heavy side effect profiles, high costs, and the resulting poor long-term patient adherence create a clear and pressing unmet medical need. The future of osteoporosis management depends on the development of a new generation of therapies that are not only effective but also safe, durable, and accessible, ultimately tilting the benefit-to-risk balance decisively in the patient's favor.

III/ Our Solution: An Encapsulated Melanin Factory

Faced with these limitations, we are developing an innovative new therapeutic approach, combining synthetic biology and biomaterials to create an in situ drug factory.

Our concept is to locally inject a hydrogel containing perfectly contained, genetically modified bacteria capable of continuously producing and secreting melanin, a molecule with powerful pro-osteogenic and antioxidant properties.[9]

Figure 2: Representation of the administration concept: local injection of the hydrogel containing the encapsulated bacteria that produce melanin at the fracture site.

The Choice of Melanin

Targeting the Root Cause of Damage during a fracture or bone injury, the microenvironment is characterized by significant oxidative stress, an overproduction of reactive oxygen species (ROS) that damage cells, provoke inflammation and reduce regeneration.

And melanin is a natural pigment with exceptional antioxidant properties. By neutralizing ROS at the injury site, it reduces inflammation, protects osteogenic stem cells, and creates a favorable environment for bone regeneration.[9]

Figure 3: Antioxidant melanin neutralizes reactive oxygen species (ROS) to reduce inflammation and promote bone regeneration.

Engineering Our Cellular Factory (E. coli W3110)

To achieve high-yield and efficient melanin production we engineered a comprehensive and robust biological system focused on both maximizing precursor availability and ensuring optimal enzyme function.

The core of our production strategy involves the surface display of the key enzyme tyrosinase Tyr1. We accomplished this by genetically fusing Tyr1 to the AIDA transmembrane autotransporter system. This fusion ensures that the bacteria synthesizes and anchors the Tyr1-AIDA complex directly onto its outer membrane. Once displayed the enzyme is strategically positioned to convert tyrosine into melanin resulting in the direct deposition of the pigment outside the bacterial cell which minimizes internal toxicity and simplifies downstream harvesting. [10]

Figure 4 : Zoom in on the mechanism of extracellular melanin production in hydrogel by tyrosinase tyr1 via the AIDA autotransporter system in W3110 Δ ompT.

To address the critical need for an abundant substrate supply we enhanced the internal metabolic flux of the melanin precursor tyrosine. This was achieved by introducing a specific point mutation S34A into the DAH7PS enzyme, a key regulator of the shikimate pathway[11]. This precise mutation effectively relieves the powerful feedback inhibition that tyrosine normally exerts on its own biosynthesis pathway. As a result the engineered strain operates without this metabolic brake enabling a continuous and accelerated production of tyrosine which is then shuttled to the cell surface for conversion.

Furthermore our system was constructed and tested in two model strains: E. coli W3110 and a genetically optimized E. coli W3110 ΔompT variant. Comparative analysis confirmed that the Δ ompT strain is significantly superior for any production of external protein [12]. This enhanced performance is directly attributed to the deletion of the ompT gene which encodes a periplasmic protease. By removing this protease our engineered AIDA-Tyr1 fusion protein is protected from degradation leading to greater enzyme stability, a higher density of functional enzyme on the cell surface and a consequent dramatic increase in total melanin output. This holistic approach of combining metabolic engineering with advanced protein display and host strain optimization ensures a highly efficient and scalable production platform.

Figure 5 : Comparison of melanin production between the wild-type strain W3110 and the optimized strain W3110 ΔompT

IV/ The promise of a Living Therapy

We believe that our project represents an interesting advance in the treatment of bone diseases, including osteoporosis, and would enable the development of safer treatments with fewer serious side effects.

Traditional treatments come with significant drawbacks but our solution is designed to overcome them. We use a localized injection to deliver the therapy directly to the site of the bone lesion. This method of local administration completely avoids the systemic effects common with oral or intravenous drugs. By concentrating the treatment exactly where it is needed we maximize its efficacy while drastically minimizing exposure to the rest of the body.

This approach leads to a theoretically superior safety profile. Conventional bone drugs are known to induce severe side effects like thrombosis, jaw necrosis and atypical fractures.[3, 5, 6] In contrast our therapy is based on melanin, a molecule already present in the body and uses perfectly contained bacteria. The result is a treatment that drastically reduces the risk of these devastating side effects.

Furthermore our system provides a sustained and continuous release of the therapeutic compound. A standard drug injection sees its concentration peak and then decrease rapidly requiring repeated doses. Our engineered bacteria overcome this limitation. They act as a permanent microscopic factory residing at the lesion site and producing the therapy in situ for the entire duration needed for complete healing.

This continuous biological production model also suggests a potentially reduced cost in the long run. While the initial development is complex the ongoing therapy is generated biologically. This could prove more economical than the repeated chemical synthesis and expensive injections of current biologic drugs.

The safety of such biosynthetic therapies is supported by emerging evidence. Although recent field approaches using live microbial therapeutics like genetically modified probiotic bacteria to treat metabolic diseases have demonstrated excellent tolerance in both preclinical models and early clinical trials.[15] Their safety relies on rigorous biocontainment strategies similar to our own system which uses multiple auxotrophies and a dependency on metabolites not available in the natural environment to ensure perfect control.

V/ Education and Human Practices

The development of a living drug requires dialogue with the public for whom it is intended. One of the central elements of our iGEM project was to proactively engage with various communities in order to educate, listen, and integrate societal considerations into our design process, and above all to listen to those suffering from osteoporosis.

We organized educational workshops with high school teachers for students and the general public at events to demystify synthetic biology, explain the principles of our project, and discuss the ethical implications of genetically modified organisms.

To conclude, our project is firmly part of this new frontier of medicine. We are creating living programmable and autonomous therapies that offer a sustainable, intelligent and safe solution to fight osteoporosis and its devastating consequences.