Overview

Cataracts, a leading cause of global blindness, primarily result from oxidative damage to lens proteins, with over 94 million people affected worldwide. Current treatment relies on invasive surgery, which remains inaccessible to many due to cost and healthcare disparities. Superoxide dismutase (SOD), a key antioxidant enzyme, neutralizes reactive oxygen species (ROS) but suffers from instability and rapid degradation, limiting its therapeutic potential.

Manganese superoxide dismutase (Mn-SOD) is an important subtype of the SOD family, with manganese ions (Mn²⁺) as an essential cofactor. This enzyme is mainly distributed in organelles with strong oxidative metabolism such as mitochondria, and is closely related to the occurrence and development of cataracts. Therefore, Our project leverages computational biology methods such as machine learning (ML) to engineer thermostable and high-activity Mn-SOD variants. These variants will be expressed in Escherichia coli BL21(DE3) under optimized conditions, and then affinity chromatographic purification and enzymatic characterization. Additionally, we also constructed a multi-scale mathematical model from microcatalytic kinetics to macroscopic 0-dimensional homogeneous system and then to 1-dimensional spatial reaction-diffusion, aiming to simulate and optimize the process of Mn-SOD eye drops from molecular mechanism to in vivo spatiotemporal efficacy, thereby significantly improving the efficiency and accuracy of formula screening and drug delivery protocol design. The Mn-SOD variant will be developed as a core ingredient in novel eye drops, providing new solutions for the prevention and early intervention of cataracts, potentially reducing the growing global burden of cataract disease (Figure1).

Figure1. The design schematic of our project.

Project target

Cataracts are the leading cause of blindness worldwide. Its pathogenesis is closely related to degeneration, cross-linking and aggregation of ocular lens proteins under long-term oxidative stress. Reactive oxygen species (ROS), such as superoxide anions (O₂⁻), are the main culprits in this oxidative damage. The eye lens has a natural antioxidant defense system, including superoxide dismutase (SOD), but its function will decline with age or under pathological conditions, resulting in an imbalance in ROS. Therefore, we boldly speculate that directly supplementing the eyes with efficient and stable antioxidant enzymes will be a promising early cataract prevention strategy. Subsequently, after interviews with ophthalmologists, graduate students, pharmaceutical companies, public questionnaires and literature research, we finally chose eye drops as our early product form to prevent cataracts. This is a comprehensive consideration of various factors such as the special physiological environment of eye diseases, the needs of preventive medicine, and patient compliance. It is a rational decision that has both targeted and applied advantages. Eye drops are a completely non-invasive, simple to operate, and can be used at home every day. This extremely high convenience is crucial for preventive programs that require long-term and daily use. It can significantly improve patient compliance, make "persistence in prevention" possible, and truly move the prevention and control window forward. If oral administration is used, the drug needs to be absorbed by the digestive system and circulated throughout the body, and finally passes through the blood-eye barrier to reach the tissues in the eye. The bioavailability is extremely low (usually <5%) and may cause systemic side effects. Eye drops adopt local administration method, allowing high concentrations of Mn-SOD to act directly on the surface of the eyeball and penetrate into the anterior chamber and lens through the corneal, greatly improving the local exposure and targeting of the drug, avoiding the first pass effect and systemic toxicity.

But tears erosion and corneal barrier in the eyes constitute unique challenges for the effect of eye drops, so our entire project design revolves around overcoming these challenges.

1. Because the eye drops stay on the eye surface for a short time, they will be diluted and removed by tears very soon. This requires that the effective substances of the product themselves must have extremely high enzyme catalytic efficiency and antioxidant capacity (high activity) in order to quickly and effectively remove ROS at limited residence time and low concentrations to achieve preventive effects.

2. Eye drop products need to maintain long-term storage stability and drug titer at room temperature. As a protein, Mn-SOD's stability directly determines the shelf life and effectiveness of the product. Therefore, we verified high thermal stability through 60°C/70°C accelerated stability experiments to ensure that our products can always remain active during shelf life and meet the basic requirements of drug regulation.

3. The production of liquid pharmaceutical preparations requires that the active ingredients must be soluble in aqueous buffer. In prokaryotic expression systems, recombinant proteins are very likely to form insoluble and inactive inclusion bodies. Therefore, we designed a molecular chaperone coexpression system and a low-temperature induction strategy. The core goal is to obtain a large amount of soluble and active Mn-SOD proteins to provide qualified raw materials for the preparation of clear, stable and efficient eye drop preparations.

4. In order to scientifically quantify and optimize our design goals, we have also built a multi-scale mathematical model, from microcatalytic dynamics to macroscopic drug efficacy simulation, providing quantitative prediction and theoretical guidance for engineering design. The microscopic model derives the rate equation based on the enzymatic reaction "ping-pong mechanism" to accurately characterize catalytic efficiency; the macroscopic zero-dimensional homogeneous model simulates the temporal dynamics of drugs and ROS through ordinary differential equations, and is used to quickly screen dose parameters; the macroscopic one-dimensional reaction-diffusion model couples space-time variables through partial differential equations to predict the permeability gradient and target exposure of drugs in the eye, ensuring the controllability and effectiveness of the dosage regimen.

Overall, our entire wet and dry experimental design forms a highly self-consistent closed-loop system designed to systematically meet all the harsh conditions of this dosage form of eye drops. Improve enzyme activity and stability through rational design and process optimization to ensure soluble production. We not only focus on mining Mn-SOD molecules with powerful biological functions, but also ensure the complete transformation path from laboratory research to industrial production through iterative simulation and verification of mathematical models. The mathematical model not only provides parameter guidance for wet experiments, but also optimizes the formulation and drug delivery frequency by simulating the spatiotemporal evolution of the drug effect in different scenarios, significantly reducing the experimental trial and error costs. Ultimately, all this effort serves a core goal: to develop a truly effective, stable, and affordable preventive eye drops that move the window for cataracts forward and reduce dependence on advanced surgery, thereby coping with the growing global burden of disease.

1 Dry experimental design

1.1 Rational design of Mn-SOD

The therapeutic application of natural Manganese superoxide dismutase (Mn-SOD) in cataract prevention is severely limited by its inherent constraints, including poor thermal stability, susceptibility to inactivation, and rapid degradation in vivo. Our project aims to overcome these barriers by employing advanced protein engineering to screen and construct Mn-SOD variants with significantly enhanced thermostability and catalytic activity. Our core strategy is a rational design approach powered by machine learning [1]. Our ML model, built on a Transformer architecture, leverages deep learning on massive evolutionary sequence data to accurately predict key residues that optimize enzymatic catalytic activity. This allows for the directed enhancement of Mn-SOD function. The most promising variants identified through this computational methods will be selected for subsequent experimental validation. Comprehensive details are provided in the Model section. (https://2025.igem.wiki/nanjing-bioX/model.html)

1.2 Mathematical Model

In order to scientifically design and evaluate the efficacy of Mn-SOD eye drops in practical application, we constructed a set of multi-scale mathematical models from simple to complex, aiming to systematically characterize and optimize the complete dynamic process of manganese superoxide dismutase (Mn-SOD) eye drops from molecular mechanism to in vivo spatiotemporal efficacy. It provides a phased and increasingly refined simulation tool for the whole process of Mn-SOD eye drops from early formulation screening to precise dosing protocol design. The system integrates three closely connected sub-models to promote simulation and prediction from three dimensions: microcatalytic mechanism, macroscopic homogeneous dynamics and spatial diffusion effect [2-4].

Firstly, in the microcatalytic kinetics model, we derive the enzymatic reaction rate equation from the first principles based on the molecular mechanism of superoxide anions scavenging by Mn-SOD and the Briggs-Haldane steady-state approximation and flow equilibrium conditions. The model quantitatively describes the dependence of clearance rate on substrate concentration and enzyme concentration from the lowest biochemical principles. Its core purpose is to establish a mathematical representation of catalytic efficiency and provide a rigorous theoretical basis for subsequent macroscopic models.

Secondly, in the macroscopic zero-dimensional homogeneous system dynamics model, we simplify the aqueous humor environment into an ideal fully mixed reactor (CSTR), and establish an ordinary differential equations (ODEs) system to simulate the change process of drug and ROS concentrations over time. This model is suitable for quickly evaluating the overall pharmacodynamic characteristics of different doses and formulation parameters, including the intensity and duration of ROS inhibition, and provides an efficient computational tool for early preliminary screening and parameter scanning.

The target that drugs for preventing cataracts need to act on is the lens. Traditional models cannot answer the core questions of "how much drug will reach the lens" and "how long will it take?" Subsequently, to further improve the physiological realism and spatial resolution of the predictions, we introduce a macroscopic one-dimensional reaction-diffusion model. The model base uses reaction-diffusion partial differential equations (PDEs) to couple time and space variables, and introduces a spherical coordinate system to accurately capture the dilution effect caused by the geometry of the eyeball. The goal is to simulate the penetration of drugs into the anterior chamber after instilling from the cornea, as well as the actual distribution and exposure history at the target (lens surface), thereby providing the ultimate basis for the precise design of dosage and frequency.

Through the multi-scale modeling strategy, we expect to significantly improve the efficiency and accuracy of Mn-SOD eye drops in formulation screening, dose optimization and protocol design, from the construction of enzyme molecular catalytic behavior to the tissue-level drug distribution full-chain and quantitatively predictable simulation framework, and lay a solid computational foundation for its transformation from basic research to practical application. Comprehensive details are provided in the Model section. (https://2025.igem.wiki/nanjing-bioX/model.html)

2 Wet experimental design

Figure2. The wet experimental design schematic of our project.

2.1 Recombinant Expression Vector Design

2.1.1 Manganese Superoxide Dismutase (Mn-SOD)

Our project obtained the original gene sequence of manganese superoxide dismutase (Mn-SOD) from the iGEM Part Registry (Part ID: BBa_K4907023). The gene was originally stored in E. coli DH10 β expression. Considering that our subsequent experiments were mainly expressed on E. coli BL21(DE3) strains, we performed a comprehensive codon optimization of the Mn-SOD gene, replacing the rare codon in the Mn-SOD gene with the preferred codon of E. coli BL21(DE3). This is not only to significantly improve translation efficiency, but also to maximize the yield and soluble expression of target proteins, which is a key foundation for the industrial production of core active ingredients in eye drops in the future and reduce manufacturing costs, ensuring that our therapies have economic viability and scale potential.

The optimized sequence was commercially synthesized to produce a high-purity linear DNA fragment, which was then seamlessly assembled with a linearized pET28a expression vector using the Gibson Assembly method, resulting in the successful construction of the recombinant plasmid pET28a-Mn-SOD. This vector contained a T7 strong promoter, a lac operator system, and an N-terminal 6×His tag, providing a critical foundation for subsequent soluble expression, affinity purification, and enzymatic characterization (Figure3).

Figure3. Construction of pET28a-Mn-SOD recombinant plasmid and gene circuit design.

2.1.2 Mn-SOD Mutation

Our project employed a Transformer-based deep learning model to analyze large-scale evolutionary data on Mn-SOD, with the goal of identifying key functional residues, cooperative site interactions, and adaptive evolutionary hotspots. Based on the computational predictions, we designed precise single-point mutations to rationally engineer Mn-SOD variants with enhanced enzymatic properties. Using the previously constructed pET28a-Mn-SOD plasmid as a template, we generated each mutant via site-directed PCR amplification with specifically designed primers (Figure4).

Figure4. Construction of pET28a-mutation-Mn-SOD recombinant plasmid and gene circuit design.

2.1.3 Molecular chaperone co-expression system

In prokaryotic expression systems, recombinant proteins tend to form inactive inclusions, which is a common challenge in the development of protein drugs. For our eye drop program, obtaining large amounts of soluble, active Mn-SOD is an absolute prerequisite for subsequent purification, formulation and functional validation. This was a key obstacle to our goal of developing liquid formulation eye drops, as the proteins in inclusions are inactive and difficult to use directly in eye drops formulations.

To address the challenge of inclusion body formation and enhance the yield of soluble manganese superoxide dismutase (Mn-SOD), we designed a robust chaperone-assisted protein folding system. We selected three distinct molecular chaperone plasmids for co-expression with the recombinant plasmids in E. coli BL21(DE3). These include pGro7, which encodes the GroES-GroEL chaperone complex; pKJE7, expressing the DnaK-DnaJ-GrpE system; and pTf16, containing the gene for trigger factor. The rational design behind employing these three chaperone systems stems from their complementary mechanisms in promoting proper protein folding under prokaryotic expression conditions. Specifically, these chaperones collectively facilitate de novo folding, suppress aggregation of nascent polypeptides, and assist in the refolding of misfolded proteins, thereby significantly reducing the formation of inclusion bodies. By mitigating misfolding and aggregation, this integrated chaperone strategy notably enhances the solubility and functional yield of recombinant Mn-SOD. This approach provided a reliable foundation for subsequent large-scale protein purification, detailed biochemical characterization, and various application experiments within our project (Figure5).

Figure5. Construction of Molecular chaperone plasmids design.

2.2 Induced Expression and Purification of Mn-SOD

In order to obtain bioactive Mn-SOD with high efficiency, we developed an induction and purification strategy using the pET28a vector expressed in Escherichia coli BL21(DE3). This approach was designed to enhance soluble expression and meet downstream application requirements. The pET28a vector was selected for its strong T7 promoter, which enabled high-level transcription, and for its encoded N-terminal 6×His tag that facilitates one-step purification via nickel affinity chromatography. This method simplified the purification process while helping to maintain enzyme activity.

To address the common issue of inclusion body formation, we optimized the induction conditions by adding 0.5 mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG) when the bacterial culture reached an OD600 of 0.6, followed by incubation at 16°C for 16 hours. This low-temperature induction slows the rate of protein synthesis, promotes proper folding, and maximizes the yield of soluble protein. The purified Mn-SOD was analyzed by SDS-PAGE, which confirmed the presence of a single band at the expected molecular weight, providing a purified sample suitable for subsequent enzymatic characterization (Figure6).

Figure6. The graph of Protein affinity chromatography.

2.3 Mn-SOD Enzyme Activity Assay

We employed the classical pyrogallol autoxidation method to quantitatively determine the superoxide dismutation activity of purified Mn-SOD enzyme. This assay operates on the principle that pyrogallol autoxidizes under alkaline conditions, generating superoxide anions (O2⁻) and a chromogenic product with strong absorbance at 325 nm. SOD inhibits this reaction by catalyzing the dismutation of O2⁻. The assay was conducted in a 96-well format using a reaction mixture containing 0.1 M Tris-HCl (pH 8.2), 1 mM EDTA, and 4.5 mM pyrogallol. The decrease in the oxidation rate was monitored spectrophotometrically at 325 nm. Enzyme activity was defined as the amount of enzyme required to inhibit pyrogallol autoxidation by 50%, and specific activity (U/mg) was calculated according to Figure7. This reliable and high-throughput method allowed us to functionally validate the effect of our protein engineering strategies, such as point mutations.

Figure7. SOD enzyme activity calculation method.U/mg = SOD enzyme activity unit (Units per milligram); ΔA325 = the auto-oxidation rate of pyrogallol; ΔA'325 = the inhibited auto-oxidation rate by the sample or SOD enzyme solution; V = the Volume of enzyme/sample solution added (mL); D = Dilution factor of the enzyme/sample solution; V1 = Total volume of the sample solution (mL); m = Mass of the sample (g); 4.5 = Total reaction volume (mL).

2.4 Mn-SOD Thermostability Assay

To quickly assess the conformational stiffness of purified Mn-SOD and predict its storage stability as a pharmaceutical component, we designed an accelerated thermal stability assay that measures the enzyme’s ability to retain function after heat challenge. Purified enzymes were diluted to 0.05 mg/mL and incubated at elevated temperatures (60°C and 70°C) for varying durations (0–60 min). By measuring its activity decay rate at high temperatures (60°C and 70°C), its shelf life under milder storage conditions can be deduced, which is standard practice in pharmaceutical formulation development and is the basis for future testing experiments under other temperature conditions (e.g., 4°C or 25°C). After heat treatment, samples were immediately cooled on ice, and residual activity was assessed using the pyrogallol autoxidation assay. The relative activity was expressed as a percentage of the initial unheated activity, enabling us to compare the stability of different variants under conditions mimicking industrial processes. This assay provided critical insights into the structural robustness of engineered Mn-SOD.

2.5 Mn-SOD Antioxidant Capacity Assay

To clarify the antioxidant capacity of Mn-SOD, we used the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method to determine the total antioxidant capacity of the sample, and the method principle was based on the stable DPPH radical at the wavelength of 515nm with a characteristic absorption peak, when the antioxidant reacted with DPPH, it would cause its fading and reduce the absorbance, and the degree of absorbance change was positively correlated with the antioxidant capacity of the sample. In the experimental operation stage, the spectrophotometer was first warmed up for 30 minutes and calibrated to 515nm wavelength, and then a blank tube (containing DPPH reagent only) and a measurement tube (containing DPPH reagent and sample to be tested) were set up according to the experimental design, and after full mixing, it was reacted at room temperature for 20 minutes to prevent light interference, and finally the absorbance value at 515nm was determined after diluting the reaction solution 3 times. In the data processing stage, the absorbance difference (ΔA = A blank - A measurement) was calculated first, which directly reflected the strength of the antioxidant capacity. Then, the ΔA value was converted to the equivalent Trolox concentration by the Trolox standard curve equation y = 1.4144x - 0.0081 (R²=0.9977), and finally the total antioxidant capacity value (calculated formula: 0.707 × (ΔA + 0.0081) / Cpr) was obtained by the Trolox standard curve equation y = 1.4144x - 0.0081 (R² = 0.9977), which expressed the antioxidant capacity of each sample protein in “μmol Trolox/mg prot”. This enables comparability evaluation between different samples.

3 Application design

Our ultimate goal is to transform the engineered Mn-SOD variant into a truly effective preventative eye drop. During our Human Practices engagements, ophthalmologists and graduate students acknowledged the therapeutic potential of our engineered Mn-SOD variant for cataract prevention. However, they also highlighted significant concerns regarding its translational application. The clinical utilization of SOD is primarily constrained by rapid clearance, poor tissue permeability, and low bioavailability, largely due to the eye’s complex biological barriers such as the corneal epithelium and the blood-retinal barrier [5-6].

To overcome these delivery challenges, our future work will focus on investigating extracellular vesicles (EVs) and liposomes as next-generation targeted delivery systems based on insights from literature review. Liposomes, as clinically established delivery vectors, can be enhanced for ocular application through surface functionalization strategies. Employing cationic lipids or specific targeting ligands can improve corneal adhesion, enhance permeability, and enable precise targeting of lens epithelial cells [7-8].

A more innovative approach involves the use of extracellular vesicles (EVs)—natural, nanoscale lipid bilayer vesicles. EVs exhibit inherent biocompatibility, low immunogenicity, superior ability to penetrate biological barriers, and high efficiency in protecting encapsulated cargo from degradation. Our engineered SOD could be effectively loaded into EVs via methods such as electroporation or through genetic engineering of parent cells for overexpression. Of particular interest are mitochondria-derived vesicles (Mito-EVs). These vesicles are naturally enriched in mitochondrial proteins and metabolites, offering a novel pathway for the targeted delivery of mitochondrial antioxidants [9-11]. Since mitochondria represent the primary source of intracellular reactive oxygen species (ROS) and SOD2 (Mn-SOD) is the key enzyme responsible for neutralizing mitochondrial superoxide radicals, utilizing Mito-EVs to deliver SOD2 or SOD2 mimetics could allow for precise targeting [12]. This strategy may facilitate the neutralization of oxidative stress at its origin, whick would present a highly promising delivery platform for cataract prevention.

4 References

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