. Description .
1. The Problems We Aim to Solve
1.1 Genetic Diseases and Gene-Editing Therapies
Since the completion of the Human Genome Project in 2003, many previously "incurable" diseases, including certain types of cancer, have been identified as genetic disorders. These diseases may arise from changes in gene sequences or epigenetic modifications. Most genetic disorders face challenges such as difficult diagnosis, high treatment costs, and a lack of effective therapies. Gene diagnosis and gene therapy offer new hope for advancing pathological research and clinical treatment. Gene-editing technology can regulate the functions of endogenous genes, providing novel solutions for curing genetic diseases[1][2].
1.2 In Vivo Gene-Editing Therapy
Our ultimate goal is to achieve precise in vivo gene editing through single or multiple administrations to alleviate or potentially cure these "incurable" diseases.
2. Why Choose "Constructing a Gene-Editing Platform" as Our Project?
2.1 Common Vectors of the Cas9 Gene-Editing System
The CRISPR/Cas9 system can perform gene editing rapidly and efficiently, and plays an important role in disease models and treatment. However, the lack of safe and effective delivery vectors limits its clinical applications.
Currently, CRISPR/Cas9 delivery methods are primarily divided into two categories: physical methods and vectors. Physical methods, such as electroporation, require highly skilled operators, are technically challenging, time-consuming, and costly, and can cause cellular damage. While market-available vectors, such as adeno-associated virus (AAV) systems, exist, they have limitations related to immune responses, off-target effects, low payload capacity, and potential carcinogenic or mutagenic risks. Non-viral vectors like gold nanoparticles, liposomes, and extracellular vesicles (EVs) also need improvements in terms of operational convenience, targeting ability, and cost-effectiveness[3].
Therefore, a delivery platform for the CRISPR/Cas9 system that balances safety, low cost, high targeting ability, and ease of operation is essential.
2.2 Advantages of Artificial EcN Outer Membrane Vesicles as a Delivery Platform
Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium that is easily engineered and has a well-established safety profile. It is widely used to treat gut-related diseases and has emerged as a preferred chassis for therapeutic applications in immune- and metabolism-related conditions. EcN has immunomodulatory properties and can enhance the host's immune defense[4][5].
3. About our project
3.1 Project Goal
The main objective of this project is to build a gene-editing system delivery platform based on the artificial outer membrane vesicles (OMVs) of EcN. This platform will be characterized by low cost, high safety, and high targeting ability.
3.2 Project Design
We use EcN as a chassis cell for genetic engineering modification, and insert the T7 RNA polymerase gene into its genome to achieve high - efficiency expression of plasmids with the T7 promoter. Transform the dual - plasmid system of the CRISPR/Cas9 RNP and targeting elements into the engineered EcN to achieve in - vivo self - assembly and synthesis of RNP. After that, produce a large number of artificial outer - membrane vesicles through physical extrusion. Design two types of elements on the surface of OMVs that are easy to change the targeting ability, and display them on the outer cell membrane for precise targeting of different cells subsequently.
Element Type I: CL7 is a modified version of the DNase domain of colicin E7, retaining its high affinity for its natural inhibitor protein, Im7. It is linked to the ice nucleation protein (InaK) to enable surface display of Im7. By expressing CL7-tagged single-chain antibodies or nanobody complexes with distinct targeting properties, precise delivery to target cells can be achieved[6].
Element Type II: A structure capable of binding to the Fc (fragment crystallizable) region of antibodies has been developed, with the Z and 4Z domains identified for their high binding affinity to the antibody's Fc domain. Both domains are linked to InaK and displayed on the outer membrane surface of EcN. Commercial monoclonal antibodies with Fc domains are selected to facilitate targeted delivery to specific cells[7].
4.Project Prospects
The precision and advancement of gene-editing technologies are crucial for the future. As the technology progresses, the importance of precise targeting in delivery systems will grow. This system offers several advantages, including being "faster and simpler than AAV" and "less toxic than chemical methods." It has the potential to be a rapid and efficient delivery tool in laboratory settings.
5.References
- Gene therapy for ADA-SCID, the first marketing approval of an ex vivo gene therapy in Europe: paving the road for the next generation of advanced therapy medicinal products EMBO Mol. Med., 9 (6) (2017), pp. 737-740
- Al-Saif, A.M. Gene therapy of hematological disorders: current challenges. Gene Ther 26, 296–307 (2019)
- Liu Kun, Liu Yanqing, Huang Nan, et al. Research Progress in the Delivery of CRISPR/Cas9 System by Extracellular Vesicles [J]. China Biotechnology,2024,44(06):1-10.DOI:10.13523/j.cb.2401053
- Wu Yanrui, Wang Shuo, Liu Chuan, et al. Research on Therapeutic Engineered Probiotics in Inflammatory Bowel Disease [J]. Chinese Journal of Gastroenterology and Hepatology,2024,33(06):646-649
- Sonnenborn, U.; Schulze, J. The non-pathogenicescherichia colistrain nissle 1917—Features of a versatile probiotic. Microb. Ecol. Health Dis. 2009, 21, 122–158
- Yin W, Wang X, Liao Y, Ma L, Qiao J, Liu H, Song X, Liu Y. Encapsulating IM7-Displaying Yeast Cells in Calcium Alginate Beads for One-Step Protein Purification and Multienzyme Biocatalysis. Front Bioeng Biotechnol. 2022 Mar 17;10:849542. doi: 10.3389/fbioe.2022.849542. PMID: 35372292; PMCID: PMC8969745
- Huang, L., Tang, W., He, L. et al. Engineered probiotic Escherichia coli elicits immediate and long-term protection against influenza A virus in mice. Nat Commun 15, 6802 (2024).
