Our Project Description

Background

Colorectal cancer (CRC) ranks as the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide. In 2022, there were over 1.926 million new CRC cases and 904,000 deaths globally, accounting for 10.0% and 9.5% of total cancer incidence and mortality, respectively. By 2040, the global CRC burden is projected to increase to 3.2 million new cases and 1.6 million deaths per year, representing a rise of 63% and 73%, respectively. In China, CRC has risen to become the second most common malignancy: approximately 590,000 new cases and 310,000 deaths were reported in 2022, with the proportion of young patients under 50 years of age increasing to 15%, significantly higher than the global average.

Although existing modalities such as chemotherapy, targeted therapy, and immunotherapy have advanced, current treatment approaches still face considerable limitations. Conventional chemotherapy (e.g., FOLFOX regimen) is associated with significant toxicity, targeted drugs (e.g., anti-EGFR monoclonal antibodies) show high resistance rates, and immunotherapy is effective in only about 5% of MSI-H patients . More critically, existing drug delivery systems commonly suffer from poor targeting, low tumor penetration, and low oral bioavailability. For example, the penetration rate of nanomedicines in solid tumors is less than 1% .

Therefore, the development of a drug delivery system that combines targeting specificity, efficient drug utilization, and low toxicity is essential to overcome current therapeutic bottlenecks in CRC and enhance the efficacy of drugs acting on difficult-to-reach targets.

Problem

While existing treatment modalities can extend the survival of colorectal cancer patients, the dose-dependent nature of therapy means that therapeutic gains are often offset by increased toxicity and enhanced drug resistance. The overall efficacy remains limited, failing to fully overcome this dilemma. Consequently, there is a pressing need for more efficient delivery methods to precisely transport drugs into cancer cells. The specific challenges are manifested in the following four aspects:

In summary, the core bottlenecks in colorectal cancer treatment are insufficient tumor targeting and high toxicity. While traditional antibodies possess targeting capability, their penetration rate in solid tumors is often below 1%; liposomes are constrained by batch variability and high cost . Therefore, there is an urgent need to develop more efficient delivery methods for precisely transporting drugs deep into tumors. Activatable Cell Penetrating Peptides (ACPPs) offer a targeted solution through an MMP-9 cleavage-de-shielding mechanism : their anionic segment inhibits transmembrane transport in normal tissues, but upon cleavage in the MMP-high TME, transmembrane transport is activated, efficiently delivering drugs into cancer cells . This ensures activation is confined to cancerous tissue, resulting in low toxicity and high specificity , thus addressing the dual challenges of "inadequate penetration" and "poor selectivity." In contrast, commonly used Cell Penetrating Peptides (CPPs) like TAT, although effective in transmembrane transport, lack tumor specificity and can lead to systemic toxicity .

Inspiration

Through our investigation, we identified the antimicrobial peptide BF2 and its engineered derivative, the cell-penetrating peptide BRⅡ. BRⅡ possesses three key characteristics :

Based on the unique properties of BRⅡ, we propose a technical strategy to engineer it into an Activatable Cell-Penetrating Peptide (ACPP). This designs a penetration delivery module for transporting therapeutic agents that otherwise cannot or have difficulty crossing membranes into intracellular targets to exert pharmacological effects. We connected BRⅡ to a negatively charged peptide segment via a matrix metalloproteinase MMP-9-sensitive linker, creating a "molecular switch" system. Concurrently, the anionic shielding significantly reduces non-specific uptake of BRⅡ in normal tissues. This design leverages the high expression of MMP-9 in the tumor microenvironment, ensuring that the ACPP is cleaved and activated specifically at the tumor site, releasing the membrane-penetrating and drug-carrying BRⅡ. This enables spatially specific drug delivery. This design further enhances specificity, successfully addressing the specificity deficiency caused by relying solely on electrostatic attraction between positive and negative charges—a deficiency that arises precisely from the charge interaction between the enriched positive charges of the peptide segment and the negatively charged gangliosides highly expressed on the surface of cancer cells. By overcoming the limitations of this single interaction mode, the biosafety of BRⅡ is effectively improved.

Furthermore, considering the long-term treatment needs for colorectal cancer, we designed a tumor-recognizing drug-releasing module for oral delivery via engineered bacteria. This aims to enable non-invasive, operator-independent administration with inherent gut targeting.

Solution

Overview

Considering the actual physical conditions of colorectal cancer (CRC) patients, our solution — the "CRC-killer in and out" KIT (a delivery toolbox) — provides two administration methods: "In and ouT"

Highlights

In and Out: Biology

No. 1

TRACER:Transmembrane Responsive Activated Cancer-targeting Engineered Release

The TRACER protein comprises BRⅡ, a sensitive peptide, and a negative charged shielding peptide. In this study, interleukin-24 (IL-24) will be selected as the cargo of the activatable cell-penetrating peptide (ACPP). TRACER and IL-24 will be connected via an F Linker to construct the fusion protein TRACER-IL24, which will be used for subsequent functional verification.

• BRⅡ Peptide:
  BRⅡ is a cancer-specific cell-penetrating peptide. Leveraging its positive charge, it preferentially engages in electrostatic interactions with gangliosides, which are highly expressed on cancer cell surfaces. This triggers lipid raft-mediated macropinocytosis, enabling efficient membrane penetration and the precise delivery of therapeutic molecules into the cytoplasm. Due to the lower density of anionic components on normal cell surfaces, the binding and internalization efficiency of BRⅡ in normal cells is significantly lower than in cancer cells. This characteristic allows BRⅡ to efficiently deliver therapeutic molecules specifically into cancer cells to exert their therapeutic effects.


• Anionic Shielding Peptide:
  Connected to BRⅡ via the MMP-9-sensitive peptide, the anionic shielding peptide segment inhibits the penetrating activity of BRⅡ, preventing non-specific activation in non-tumor environments.


• Sensitive Peptide:
  The PLGLAG sequence can be specifically cleaved by matrix metalloproteinase-9 (MMP-9). Based on this, through literature research combined with bioinformatics analysis, we found that the expression level of MMP-9 in colorectal cancer tissues is significantly higher than that in normal tissues, suggesting that MMP-9 has the potential to serve as a molecular marker for colorectal cancer.


• F Linker:
  The F Linker is a flexible connecting sequence used to link two proteins, providing structural flexibility and functional cooperativity.
• IL-24 Protein:
  IL-24 serves as the therapeutic drug molecule, possessing anti-cancer activity... Consequently, it achieves efficient, low-toxicity, multi-target, and broad-spectrum anti-cancer effects.
  1. It induces the expression of GADD family genes, phosphorylates eIF2α, activates caspase cascades, and induces apoptosis.
  2. It upregulates Bax/Bak, inhibits Bcl-2/Bcl-xL, promotes cytochrome c release, and induces mitochondria-dependent apoptosis.
  3. It blocks PI3K/AKT, STAT3, and Src signaling, inhibits VEGF/TGF-β, thereby suppressing tumor angiogenesis, cancer cell proliferation, and metastasis.
  4. It stimulates T/NK cells to secrete IFN-γ and TNF-α, enhancing immune recognition.
  Consequently, it achieves efficient, low-toxicity, multi-target, and broad-spectrum anti-cancer effects against tumor cells.

BRⅡ possesses the ability to carry drugs into cells, relying on its positive charge to bind to cell membranes. The negatively charged shielding peptide inhibits the non-specific adsorption of BRⅡ in non-cancerous environments. The sensitive peptide is cleaved upon encountering MMP-9, which is highly expressed in the tumor microenvironment. IL-24 is connected to BRⅡ via a Flexible Linker. When the sensitive peptide is cleaved, the negatively charged peptide detaches, allowing BRⅡ to precisely deliver IL-24 into cancer cells, inducing their apoptosis.

No. 2

Deliver

In the intestinal tract of colorectal cancer patients, the level of oxidative damage is significantly elevated, primarily due to massive infiltration of inflammatory cells and mitochondrial dysfunction, leading to excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These oxidants can directly attack DNA, inducing gene mutations. Based on this, we propose the:

This module utilizes the SOS promoter to sense DNA damage, activating the SRRz lysis protein to release the drug, while employing EGFP for visual monitoring of the process.

• Activation (Activator by DNA Damage):
  In the high oxidative stress microenvironment of colorectal cancer, DNA damage products accumulate significantly. Therefore, we introduce the SOS response mechanism derived from E. coli: an inducible DNA repair system in E. coli that is activated when DNA is damaged. When bacterial DNA is damaged, the RecA protein is activated and mediates the auto-proteolysis of the LexA repressor, thereby derepressing the SOS promoter. The expression level from the SOS promoter increases, subsequently activating the downstream LuxI gene to express the acyl-homoserine lactone (AHL) synthase, completing the conversion from DNA damage to a biomolecular signal.


• Lysis:
  When the intracellularly synthesized AHL accumulates to a certain threshold, it binds to the constitutively expressed LuxR protein, forming a LuxR-AHL complex. This complex specifically activates the Lux promoter, thereby driving the expression of the downstream lysis gene SRRz. SRRz encodes three lysis proteins: the holin encoded by the S gene forms pores in the cell membrane; the endolysin encoded by the R gene hydrolyzes the peptidoglycan layer of the cell wall; and the Rz gene product cleaves the linkages between the peptidoglycan and the outer membrane. These three proteins act synergistically to thoroughly disrupt the cell structure, ultimately leading to bacterial lysis.Once the to-be-lysed proteins are inactivated, the remaining bacterial population continues to grow and repeats the aforementioned process, forming an oscillatory drug delivery effect.


• TRACER-IL24::
  An efficient fusion protein for anticancer therapy designed in this study comprises a TRACER domain and interleukin-24 (IL-24).


• Reporter:
  The Lux promoter activates the expression of the EGFP reporter gene, used for characterizing the growth and lysis status of the engineered bacteria in vitro.


• Therapeutic:
  Throughout this process, the strong constitutive J23100 promoterhttps://parts.igem.org/Part:BBa_J23100 continuously drives the expression of the therapeutic protein molecule IL-24. IL-24 is a protein molecule capable of inducing apoptosis in cancer cells. IL-24 is connected to TRACER via a Flexible Linker (Flinker), achieving efficient and precise anti-cancer functionality. This biological circuit design implements a comprehensive process of DNA damage detection and response, coupled with bacterial lysis and drug release.

Our TRACER and Deliver Module and Tumor-Sensing Drug Release Module work in concert to execute a coordinated "recognition-release-penetration-killing" process targeting cancer cells.

After the engineered bacteria reach the tumor site, the Tumor-Sensing Drug Release Module captures DNA damage signals caused by high oxidative stress via the SOS promoter. This activates the expression of the LuxI gene, leading to the synthesis of AHL. AHL then binds to the LuxR protein, forming a tetrameric complex that subsequently activates the Lux promoter. This activation drives the expression of the SRRz lysis proteins, resulting in bacterial lysis and the release of the IL-24 prodrug, which is constitutively expressed by the J23100 promoter.

Following drug release, the Activatable Cell-Penetrating Peptide (ACPP) delivery module takes over: BRⅡ, with its positive charge, specifically recognizes the relatively high abundance of gangliosides on the cancer cell surface. Concurrently, the anionic shielding peptide is linked to BRⅡ via an MMP-9-sensitive peptide linker. When matrix metalloproteinase-9 (MMP-9) activity is elevated in the microenvironment, the sensitive linker is cleaved, the shielding peptide dissociates, and BRⅡ is activated. The activated BRⅡ then carries IL-24 across the membrane into the cytoplasm. Inside the cell, IL-24 triggers multi-pathway synergistic apoptosis, achieving multi-target, efficient, and low-toxicity killing of the cancer cells.

No. 3

Technical Route Flowchart for Oral Bacteria

• TRACER:
  First, oral enteric-coated capsules with a calcium alginate shell are administered. These capsules gradually disintegrate in the intestine, releasing the engineered bacteria. These bacteria then colonize the intestinal mucosa and initiate the Tumor-Sensing and Drug Release Module. This module senses DNA damage within the high oxidative stress microenvironment of colorectal cancer via the SOS promoter, activating LuxI gene expression to produce AHL synthase. When AHL accumulates to a certain threshold, it binds to LuxR, triggering the Lux promoter to drive the expression of the SRRz lysis proteins, leading to bacterial lysis. Consequently, the IL-24 drug, constitutively expressed by the J23100 promoter, is released. Simultaneously, EGFP produces green fluorescence, allowing us to monitor the drug release status during in vitro experiments.


• Deliver:
  Subsequently, the Penetration and Delivery Module comes into effect. The TRACER protein contains BRⅡ, the sensitive peptide, and the anionic shielding peptide. Within the tumor microenvironment, MMP-9 protease cleaves the sensitive peptide, causing the anionic shielding peptide to detach and thereby releasing active BRⅡ. Leveraging its positive charge, BRⅡ binds to the negatively charged gangliosides on the cancer cell surface, precisely delivering IL-24 into the cancer cell interior. Inside the cell, IL-24 triggers cancer cell apoptosis by modulating the synergistic effects of multiple signaling pathways, achieving highly efficient and low-toxicity anti-cancer activity.


• Lysis Cycle:
  on our module, the engineered bacteria will exhibit a periodic oscillatory behavior of alternating growth and lysis in the intestinal tract. Within the patient's daily physiological rhythm, the interval between two meals serves as the core therapeutic window. During this period, the engineered bacteria undergo multiple rounds of lysis, releasing drugs continuously in a "pulsatile" manner to maintain therapeutic pressure on cancer cells.Meanwhile，the intestinal environment renewal (e.g., intestinal mucosa shedding and renewal) triggered by the patient's eating behavior will automatically terminate the current treatment cycle and clear the colonized engineered bacteria. This not only effectively prevents excessive proliferation of the bacterial population but also completes the "clearance" of colonization sites simultaneously, preparing for the implantation of the next batch of engineered bacteria and the initiation of a new round of treatment cycle, thereby ensuring the safety and sustainability of long-term treatment.

In and Out: Hardware

To address the challenges of patient acceptance and precise drug release, our hardware team developed a microneedle-based smart drug delivery system. Microneedle patches consist of tiny, dissolvable needles that painlessly penetrate the skin and release therapeutic proteins directly into the bloodstream, achieving higher delivery efficiency than traditional oral methods. To enable precise control over the release process, we integrated flexible electronics—including a heating film, PCB board, and smartphone-controlled circuits—into the patch. This design allows us to fine-tune the release rate and profile, supporting programmable modes such as rapid, delayed, or sustained delivery. The system can be used independently, offering a patient-friendly alternative to engineered bacteria, or in combination with them to provide adjustable therapeutic boosts when needed. By bridging synthetic biology with intelligent hardware, we created a dual therapeutic platform that is effective, controllable, and patient-centered.

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