Abstract

Hemorrhoids present significant treatment challenges such as symptomatic relief, surgical pain, and high recurrence rates. Based on sociological research, we developed "HemorrEaser", a synthetic biology therapy validated through wet and dry lab experiments. Using engineered E. coli Nissle 1917 in oral and topical formulations, our design features three innovations: OMV-delivery bioPROTAC-mediated HIF-1α degradation combined with an activatable anti-VEGF nanobody to suppress pathological angiogenesis; smart release of anti-inflammatory peptides via ROS-responsive mechanisms for rapid pain relief; and a dietary-component-responsive riboswitch enabling medicine-food collaboration for treatment regulation and safety control. HemorrEaser provides a precise, minimally invasive therapeutic strategy while establishing a new chronic disease management paradigm through lifestyle-integrated therapy.

Background

Hemorrhoids are a common yet frequently overlooked condition. In China, the popular saying "nine out of ten people suffer from hemorrhoids" may be exaggerated, but epidemiological studies confirm that nearly half of adults worldwide experience them during their lifetime.[1]

Despite this prevalence, research remains limited due to mild and recurrent symptoms, social stigma, and widely varying severity—factors that often relegate such conditions to lower research and funding priorities.[2] Patient awareness and treatment-seeking are also suboptimal, with many avoiding medical consultation out of embarrassment. Conventional treatments offer only temporary relief, while surgery—though effective in severe cases—is painful, slow to heal, and prone to recurrence.[3]

In response, our team aims to introduce synthetic biology into hemorrhoid care by developing precise, personalized therapies and exploring a new synergy between treatment and daily life through "Medicine-Food Collaboration".

Our Solution

Our project utilizes E. coli Nissle 1917 (ΔnlpI) as the chassis organism. When administered as an oral medication, it secretes bacterial outer membrane vesicles (OMVs) to function as a delivery module, transporting the anti-angiogenesis module. [4,5,6] When patients use the topical version of our product, the engineered bacteria within can sense the reactive oxygen species (ROS)[7] indicative of inflammation in hemorrhoidal tissue and locally secrete engineered melittin[8]. We expect that HemorrEaser will not only halt the progression of hemorrhoids and promote healing but also alleviate the pain associated with hemorrhoidal inflammation. Additionally, we employ a riboswitch[9] that responds to a specific metabolite found in certain foods. This serves to prevent bacterial leakage, provides a method to cease treatment, and acts as an interface for our "Medicine-Food Collaboration" approach.

With HemorrEaser, we aim not only to develop a broader synthetic biology-based treatment for hemorrhoids but also to assist patients in cultivating healthier dietary habits through this medication.

• Anti-angiogenesis Module: We focused on the significant roles played by HIF-1α-mediated angiogenesis under hypoxic conditions and the VEGF signaling pathway in the development and progression of hemorrhoids. [10,11,12,13] Therefore, we designed a bioPROTAC that specifically binds to and mediates the ubiquitination and degradation of HIF-1α under hypoxia.[14,15] Concurrently, we use an anti-VEGF nanobody to inhibit angiogenesis. To mitigate potential side effects of the anti-VEGF nanobody, we engineered it into a probody that is specifically activated by matrix metalloproteinase-3 (MMP3) at the hemorrhoid site. This further enhances the safety profile of HemorrEaser.
• Anti-inflammatory Module: HemorrEaser secretes engineered melittin (NKU-China, 2024), which functions as an anti-inflammatory protein, under the induction of ROS. Aided by the intrinsic inflammatory tropism of E. coli Nissle 1917, the melittin can better reach the inflamed hemorrhoidal tissue to suppress inflammation and alleviate patient pain.
• Suicide and Medicine-Food Collaboration Module: HemorrEaser utilizes a riboswitch that negatively regulates downstream gene expression in response to a specific compound derived from food. In the absence of the ligand, the engineered bacteria initiate expression of the CcdB toxin protein[16], leading to self-elimination and cessation of therapy. To meet specific requirements of some stakeholders during the treatment cycle, we incorporated an "expiry-date" circuit that delays bacterial lysis[17], allowing the engineered bacteria to remain active for extended periods without frequent dosing. In the topical application mode, the engineered bacteria contain only a simple negatively regulated riboswitch, enabling a rapid response to inflammation and ensuring quick elimination after the task is completed.
• Delivery Module: For the bioPROTAC to function from the intestine to the anal region, we use OMVs as the delivery vehicle.[18] These OMVs are modified by displaying an OmpA-CAR fusion protein on their outer membrane, targeting neo-vascular sites in hemorrhoids[19]. This approach helps reduce potential off-target effects and improve the safety of HemorrEaser.[20]

Figure 2. Functional modules involved in HemorrEaser.

(A)Global control module; (B)Anti-inflammatory module; (C)Anti-angiogenesis module; (D)Suicide(right) and Medicine-Food Collaboration(left) module.

1. Oral Formulation: This primarily contains the anti-angiogenesis module, the Medicine-Food Collaboration module, and the delivery module. It is intended for the long-term treatment and suppression of hemorrhoids, while simultaneously assisting patients in developing healthier eating habits through the Medicine-Food Collaboration mechanism. We propose using enteric-coated capsules to deliver the engineered E. coli Nissle 1917 (ΔnlpI) to the gut for colonization, establishing the basis for "Medicine-Food Collaboration".[21]
2. Topical Formulation: This mainly consists of the anti-inflammatory module and the suicide module, designed for emergency management during flare-ups of hemorrhoidal inflammation. We propose an ointment containing TPP(Thiamine Pyrophosphate) and the engineered bacteria as the specific form for topical application, aiming to achieve a rapid response to inflammation and ensure swift elimination of the bacteria post-task.

Figure 3.The two administration routes of HemorrEaser.

Innovation

Unlike traditional treatments, HemorrEaser targets the core pathological process of hemorrhoid formation – pathological angiogenesis. The project innovatively combines a bio-based proteolysis targeting chimera (bioPROTAC) with an anti-vascular endothelial growth factor (VEGF) nanobody: the bioPROTAC selectively degrades key pro-angiogenic target proteins, while the anti-VEGF antibody blocks the core signaling pathway. This intervention at the etiological level not only significantly enhances therapeutic efficacy but also effectively improves treatment precision, thereby achieving the goal of precision medicine.

Building on effective treatment, HemorrEaser establishes a "Medicine-Food collaboration" regulatory paradigm through a simple design concatenating a riboswitch with a toxin protein coding sequence. This innovatively integrates the traditional chinese medicine concept of "preventive treatment" into modern therapy, incentivizing healthy eating habits to achieve dynamic synergy between treatment and disease prevention or recurrence control. Significant efforts in experimentation, modeling, and human practices were dedicated to refining this paradigm.

To address clinical diversity, the project developed both oral and topical formulations. The oral formulation supports long-term systemic treatment and is key to realizing "Medicine-Food Collaboration"; the topical formulation provides rapid local relief. This design aims to optimize patient compliance and coverage of practical application scenarios.