Team:UCL/Composite Part

Best Composite Part

Improved Part from the UCL iGEM 2019 team

BBa_K3111502 - T. maritima encapsulin monomer fused to DARPin929 and co-expressed along miniSOG with a targeting peptide.

This composite part formed our drug delivery vehicle and was used to demonstrate its proof of concept application in the potential treatment of HER2 positive cancer. This involved cloning and expression of the assembled encapsulins with surface displayed DARPin929, followed by downstream cloning of the photosensitiser miniSOG with a C-terminal tag (1) which allows cargo loading. We proceeded to study targeted delivery and cytotoxicity on SK-BR-3 cells which overexpress HER2 receptors, to which the fused DARPin929 is specific (2,3). This involved, incubating the sample with cultured SK-BR-3 cells and illuminating them in order to activate miniSOG's generation of singlet oxygen. Taking advantage of the fluorescent properties of miniSOG the cells were observed under the confocal microscope to observe uptake. After imaging imaged the cells subjected to flow cytometric analysis to determine the pattern of cell death and overall loss of viability. These experiments show that our drug delivery platform successfully targets HER2 overexpressing SK-BR-3 cells and kills them at a significantly higher degree than MSCs once illuminated with light, thus demonstrating double selectivity in terms of cytotoxicity. We believe this composite part has proven the concept of Encapsulin-DARPin drug delivery platform, and it can be further adapted upon by using different encapsulins, DARPins and cargo.

References

  1. Cassidy-Amstutz, C., Oltrogge, L., Going, C., Lee, A., Teng, P., Quintanilla, D., East-Seletsky, A., Williams, E. and Savage, D. (2016). Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin. Biochemistry, 55(24), pp.3461-3468.
  2. Siegler E, Li S, Kim YJ, Wang P. Designed Ankyrin Repeat Proteins as Her2 Targeting Domains in Chimeric Antigen Receptor-Engineered T Cells. Hum Gene Ther [Internet]. 2017 Sep 1 [cited 2019 Sep 14];28(9):726–36.
  3. Steiner D, Forrer P, Plückthun A. Efficient Selection of DARPins with Sub-nanomolar Affinities using SRP Phage Display. J Mol Biol [Internet]. 2008 Oct 24 [cited 2019 Sep 14];382(5):1211–27.