Team:iBowu-China/Poster

Poster: iBowu-China



Sureloc - Surely Locates Cancer
Presented by Team iBowu-China 2020

Mika Yokota¹, Angela Zheng¹, Wendy Zhang¹, Lucas Ji¹, Atom Wang¹, Linda Geng¹，Angela Zhang¹, Zhe Leng²,Ivan Zhao³,Lingyan Wang§

¹iGEM Student Team Member,²iGEM Team Mentor,³iGEM Team Primary PI,§Faculty Sponsor, School of Life Sciences, Peking University, Beijing, CN

Abstract

Non-small cell lung carcinoma (NSCLC) is a common type of epithelial cancer, which is not sensitive to chemotherapy or radiotherapy. To address this issue, our team developed a therapy based on magneto-hyperthermia. We designed a vector that codes for magnetic protein crystals (MPCs) through the use of a tumor-specific promoter. Liposomes with surface PEG-GE11 modifications carrying MPC vectors will target NSCLC cells. Post transfection, targeted tumor cells will express MPCs, which enable the binding of iron. Iron-loaded MPCs will then respond to localized alternating magnetic fields and induce cell death. The active targeting ligand, tumor-specific promoter, and localized alternating magnetic fields provide triple targeting security. Currently, we completed the in vitro isolation and purification of the MPCs with successful confirmation of their magnetism. The successive steps are to control the MPCs’size, add the internal targeting ligand, and experiment the targeting capabilities in vivo.
Project Goals
We postulate that an engineering protein could be assembled for magnetic sensing and generate substantial magnetocaloric effect. With these features, this protein is an alternative material for magnetic hyperthermia. Moreover, it should be targeted to the tumor cells for the specific therapy.

Triple Targeted Cancer Therapy

  1. targeted gene circuits by tumor-specific promoter.
  2. targeted delivery of liposomes through surface modification.
  3. targeted tumor elimination by localized alternating magnetic field.

A Universal Basis For Substitution

The same magnetic protein sequence can be matched with different tumor-specific promoters, surface modifications, and location of the localized alternating magnetic field to target other types of cancers.
What is Non-Small Cell Lung Carcinoma？

Fig 1. Non-small cell and small cell lung cancer

Lung cancer can be divided into two main pathological types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for more than 80% of the total number of lung cancers, including squamous cell carcinoma, large cell carcinoma, adenocarcinoma, and other histological types (Torre L et al.,2015).

Reference
1. Torre, L. A. et al. Global cancer statistics, 2012. CA Cancer J. Clin. 65, 87–108 (2015)
Current Treatments of NSCLC

Fig 2. Current treatments of non-small cell lung cancer

The current treatments for advanced NSCLC are mainly chemotherapy, radiotherapy, targeted therapy, and immunotherapy. The advent of targeted therapy and immunotherapy has brought NSCLC treatment into the era of "individualized therapy" and "precision therapy”(Herbst R S et al.,2018).

Reference
Herbst R S , Morgensztern D , Boshoff C . The biology and management of non-small cell lung cancer[J]. Nature, 2018, 553(7689):446.
Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, 543–550 (2014)
Design
Our Solution-a targeted therapy based on magneto-hyperthermia
Fig3. A targeted therapy based on magneto-hyperthermia
Hyperthermia is one of the oldest therapies known,  used as an anti-cancer therapy where the tumor temperature is increased to kill cancer cells.However, toxicity due to the similar responses of malignant and healthy tissues to hyperthermia remains a barrier to clinical application. To make our therapy safer with high specificity, we choose to use an engineered biodegradable magnetic protein crystal as the heating agent with triple targeting security.
Build

Core Component - Magnetic Protein Crystal


Fig4.The structure of magnetic protein crystal

  • Ferritin: naturally capable of mineralizing iron.
  • Ibox-PAK4cat: catalytic domain of PAK4 kinase binds with Inka1 and further cause the formation of crystals.
  • IBox-PAK4cat-Ferritin: formation of this rod-like magnetic protein crystal
  • More than 10 million of ferritin subunits can bind to its hollow channel for iron storage.

    Target Delivery System Based on Liposome

  • For nanoparticle targeting delivery, liposome is modified with GE11.
  • For the immunological recognition, PEG is also used for the modification.
  • For further tumor-specificity, the lung cancer cell high expression promoter is designed into the vector.
    • Improved Targeting and Safety
    Based on the primarily experimental results (figure 1 in result) and investigation, changes were made in our design.
  • The magnetic crystals were isolated and iron loaded in vitro before delivery.
  • Magnetic proteins were delivered by the ADC (antibody-drug conjugate).
  • The magnetocaloric effect was control by the area of the localized alternating magnetic field
    • Results
    Fig1 a) Wt-PAK4 and b) Ft-PAK4 expression results in intracellular crystals
    Fig2 Crystals isolated from cells. a) Wt-PAK4 crystals, b) Ft-PAK4 crystals.
    Fig3 Prussian Blue (PB) staining rusults: a) Wt-PAK4 crystal, b) Ft-PAK4 crystals.

    Fig4 Magnetic response of Ft-Fe crystal.

  • Wt-PAK4 (with GFP) and Ft-PAK4 crystals grow a single needle-like crystal in HEK293 cell (Fig1). However, fail to be iron loaded in vivo.
  • Crystals successfully isolated from cells (Fig2).
  • The Ft-PAK4 (b) crystals turned a deep blue color in Prussian blue (PB) staining after iron loaded (Fig3).
  • Protein crystals showed the magnetic response under different lift height (Fig4).
    • Summary
  • We have engineered Ft-PAK4, a genetical protein crystal containing ferritin that grows inside mammalian cells.
  • We have isolated and characterized isolated Ft-PAK4 crystals and Wt-PAK4 crystals.
  • We have detected magnetic forces when PAK4 crystals loaded with iron in vitro. These protein crystals are attracted to external magnetic field and move toward magnets.
  • We have presented regimens in order to realize the magnetic control of genetic targeting in vivo in the future therapy.

    Further work

    Targeting problem

    For further magnetic crystal application, two therapy regimens are designed:
    1) a modified liposome delivery system or 2) conjugated with antibodies.
  • Through encapsulating MPC in liposomes, magnetic liposomes offer multiple functions for biomedical applications.
  • Antibody-drug conjugates (ADCs) allow an antibody to be conjugated to MPC via a linker which is delivered to its unique target.

    Specific expression problem

    A tumor-specific expression promoter will be used for the specific expression.
    • Human Practices

    STAGE 1: Preliminary Research

    To establish a set of success criteria for our product

    Literature Review:Potential solutions
    Patients Interview:Limitations of current treatments,Expectations for future treatments
    VC Investors Interview:Demand of the market

    STAGE 2: Project Modification

    To assess the validity of the theoretical foundation

    Professor Dong,Aarhus University
    ” Magnetic protein crystals may be unable to load sufficient iron in vivo.”

    Professor Xie,Chinese Academy of Sciences
    ”Ferritin may be incapable of storing enough iron. Transferrin and magnetosomes are other potent candidates. ”

    STAGE 3: Project Specification

    Move towards the implementation of the plan

    We interacted with
    2 Doctors,2 Scientific Researchers,2 Pharmaceuticals: BeiGene ＆ 3D Medicine

    Discussed the following
    Immune Response,Temperature Control,Targeting Ability,Internalization Issue
    Modeling

    We built a two-dimensional stationary fluid flow with liposomes in it. A straight charged wire is added outside. By building and calculating the differential equations that described the liposomes' trajectory in the fluid flow, we concluded that the number of captured liposomes in the targeted area increase when an external alternating magnetic field is added.

    With magnetic field
    Without magnetic field
  • Targeted area is shown in RED.
  • Count liposomes that pass through targeted area.
  • When external alternating magnetic field is added, liposomes are attracted to the targeted area.
    • Acknowledgements
    Professor MD Dong
    Professor Can Xie
    Lingyan Wang
    Xiaomei Lin
    Zhe Leng
    Ivan Zhao