In brief • Current therapies fail to effectively treat cancer patients, largely due to the difficulty in distinguishing between healthy and cancer cells • We designed a CRISPR-based synthetic system that will be assembled and activated only in cancer cells • Proof-of-concept results show that the Boomerang system is highly selective and does not affect healthy cells • The system can be easily adapted for every patient and tumor using the individual tumor characteristics Klac BD Mutant 20,000 people die of cancer every day Our system Results Fig. 2. eGFP and mCherry expression, under human TERT and survivin promoters, respectively, in fibrosarcoma cancer cells (upper panels) vs. healthy fibroblasts (lower panels), using calcium phosphate plasmid transfection. Results show specific expression in cancer cells. This collaboration can take the Boomerang system to the next level of cancer detection&diagnostics. Selective expression of the Affibody in cancer cells can synergistically increase both systems` potential. Human Practices Culture and education School workshop Educational convention • Increased accessibility to information about synthetic biology by arranging unique events • Establish a dialogue with industry experts to improve our project • Successful collaborations with iGEM teams • Collaboration and advice panels with leading experts in cancer research, synthetic biology, and bioethics Ethics and philosophy Ethical aspects of CRISPR use Arranged panels Experts from the fields of basic and clinical cancer research and synthetic biology Special meetings Ministry of Health Directors of the Oncology Dept. at Soroka Medical Center and Sheba Cancer Research Center Public outreach Radio , Video, Media, Social networks: FB, Twitter Thousands of followers External website Science on the Bar event Conventions Collaboration China-Amoy, article France-Aix-Maseille, Israel-Technion, convention Entrepreneurship Meeting with synthetic biology companies Meeting with Venture Capital Crowd funding Future vision The third part of the Boomerang system is a construct that, upon activation, drives the expression of any gene of interest for multiple applications: 1. Diagnostics through a secretion of protein biomarker 2. Visualization of the tumor for surgical removal 3. Inducing apoptosis/cell death specifically in cancer cells The Boomerang system will allow easy configuration of the three-plasmid CRISPR-based gene activation to adapt the treatment to every tumor and patient. This way side-effects could be minimized. Moreover, constant adaptation of the designed constructs will ensure the effectiveness of the system, despite the tumor heterogeneity. Cancer-specific expression of gRNA and Cas9 will activate a third construct containing a synthetic promoter that can drive the expression of any target protein One Boomerang - Three Applications Motivation Fig. 1. hTERT and Survivin expression levels in several human cancer cell types, evaluated by qPCR. The results confirm the suitability of these two promoters for Boomerang system. Fig. 3. Left panels: Similar AAV transduction efficiency in cancer and healthy cells. Right panels: Viral transduction of eGFP and mCherry under human TERT and survivin promoters, showing specific expression in cancer cells. Collaboration “Master” template – for modular cloning Synthetic activation promoter RGR design for gRNA – Ribozyme-gRNA-Ribozyme Design Highlights Research Aim phTERT pSurvivin Acknowledgments Lab Workflow Fig. 4. The activation of the entire Boomerang system. The three-plasmid CRISPR-driven eGFP expression is shown exclusively in cancer cells (upper panels), vs. no detected signal in health cells (lower panel). • To design a modular system that will transform the tumor into a in-cell drug factory • To experimentally validate the 3- component CRISPR-based Boomerang gene activation system in human cancer cells Stockholm team - Affibody approaches Confocal imaging of Affibody expression in human fibrosarcoma – BGU team