(G-250) Tuning Artificial Cytoskeleton of Cyborg Probiotics for Breast Cancer Gene Therapy

Introduction:: Breast cancer affects 1 in 8 women worldwide. Despite the available treatments, such as chemotherapy and radiation therapy, tumor eradication is often unsuccessful, leading to relapse and metastasis. Targeted therapies, such as CRISPR-Cas9 gene therapy, emerge as promising therapeutics to address the specific molecular roots of the disease. However, current methods to deliver CRISPR-Cas9 to breast cancers (e.g., viruses and nanoparticles) have significant limitations in the penetration of solid tumors and molecular size of CRISPR-Cas. To maximize the efficacy of cancer gene-therapy, a sufficient amount of multi-targeting Cas9 and sgRNA must be delivered. A potential solution to the delivery problem is bacteria. They move by active transport and can deliver large cargo, but they risk contaminating non-targeted sites in a patient. Here, I create nonreplicating-but-active Cyborg Bacteria that are tuned with different hydrogel components for breast-cancer gene-therapy. Cyborg Bacteria are hybrid material-cell entities created by the infusion of a synthetic polymer network inside the bacteria, rendering them incapable of dividing, yet preserving key essential functions, including cellular metabolism, motility, protein synthesis, and compatibility with genetic circuits (Contreras-Llano, et al.). Cyborg Bacteria’s non-replicating-but-active feature enables the safe and superior delivery of gene therapy constructs to breast cancers.


Materials and Methods:: New components, including 4-arm-PEG-Acrylate, Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and modified dsDNA sequence were used to tune the artificial cytoskeleton of Cyborg Bacteria. To create nonreplicating Cyborg Bacteria, bacteria were incubated with hydrogel buffer containing these components and flash-frozen in cold methanol, followed by incubation at -80°C. After thawing and recovery in fresh media, UV irradiation was used to crosslink the hydrogel inside the bacteria. Cyborg Bacteria were then washed with PBS and resuspended in appropriate media (Contreras-Llano, et al.). To report successful hydrogelation of different component-tuned Cyborg Bacteria, hydrogel buffer contained fluorescein diacrylate that crosslinked with PEG and acted as a reporter for intracellular hydrogelation. Flow cytometry analysis and fluorescence microscopy imaging were used to detect the fluorescein signal from Cyborg Bacteria across multiple days. To further measure the metabolic activity of Cyborg Bacteria, CTC RedoxSensor dye was used to stain the cells. Cells were then analyzed using flow cytometry and fluorescence microscopy imaging. Non-replicating phenotype of FACS sorted Cyborg Bacteria was confirmed by colony-forming-unit assays. To validate the CRISPR-Cas9 knockout of a model gene and ERBB2 oncogene, sgRNAs were designed to cleave the genes in vitro and target cells.


Results, Conclusions, and Discussions:: I have generated Cyborg E. coli Nissle 1917 that maintains high metabolic activity and a nonreplicating phenotype over three days, as measured by flow cytometry and live cell counts (Fig. 1B). Different components of the intracellular hydrogels generate differing degrees of impact on the non-replicating and metabolic activity of the Cyborg bacteria. Specifically, Cyborg Bacteria tuned with 2-arm PEG and LAP photoinitiator show increased hydrogelation efficiency, while Cyborg Bacteria tuned with a larger synthetic polymer 4-arm PEG with both PA and LAP photoinitiators show similar degrees of hydrogelation efficiency and metabolic activity to our conventional Cyborg Bacteria protocol. FACS-sorted Cyborg bacteria modified with different hydrogel components have demonstrated a purity of over 99.999%, making them safe and effective for therapeutic use (Fig. 1C). Furthermore, the successful invasion of WT and Cyborg E. coli into HEK293 cells is shown in confocal microscopy (Fig. 1D). Both WT and Cyborg E. coli were colocalized in the cytoplasm of HEK293 cells, while non-invaded bacteria were removed with gentamicin for qPCR quantification of intracellular bacteria. E. coli carrying a reporter EGFP gene in plasmids shows the successful transfer of the plasmids and expression of GFP in HEK293 cells (Fig. 1E). Furthermore, we design and validate CRISPR-Cas constructs that knockout a target gene in vitro and inside the HEK293 cells. The sgRNAs in the CRISPR-Cas construct were carefully designed for their specificity, low off-target activity, and high efficiency against the target genes. Our designed CRISPR-Cas constructs cleave the model gene and ERBB2 oncogene successfully both in vitro and in cells. The cyborg E. coli are then investigated for their efficacy in the CRISPR-Cas-therapy of BT474 and SKBR3 breast cancer cells.

In conclusion, I have shown that Cyborg Cells are easily tunable with different hydrogel components. Furthermore, I have tested the ability of bacteria in invading and delivering cargo to mammalian cells. My Cyborg-cell based approach leverages the bacteria's ability to target solid tumors while minimizing the risk of contamination in non-targeted sites. Overall, these results demonstrate Cyborg Bacteria's potential as a CRISPR-Cas9 gene editing delivery system for breast-cancer therapy.


Acknowledgements (Optional): : I would like to thank NIGMS and NCI for supporting the project. I would also like to thank the members of Tan and Hovey lab for their advice and support.


References (Optional): : Contreras-Llano, L. E., Liu, Y.-H., Henson, T., Meyer, C. C., Baghdasaryan, O., Khan, S., Lin, C.-L., Wang, A., Hu, C.-M. J., Tan, C., Engineering Cyborg Bacteria Through Intracellular Hydrogelation. Adv. Sci. 2023, 2204175.