Engineering Microglia for Neural Cell Therapy LETHBRIDGE BRAINIACS: Dennis Bettenson Rhys Hakstol Harland Brandon Suneet Kharey Evan Caton Kelsey O’Brien Rachael Chan Dustin Smith Billy Cowitz Zak Stinson Aubrey Demchuk Scott Wong Graeme Glaister Hans-Joachim Wieden
Background How many of you know someone who has been affected by: Traumatic Stroke Brain Injury (TBI)
Background • 1.5 million people experience a TBI each year in the USA and 800,000 suffer from a stroke • Incidence of TBI is higher than multiple sclerosis (MS), spinal cord injury, HIV/AIDS, and breast cancer combined • Combined health care costs of stroke & TBI estimated to be $110 billion in the USA alone
Policy and Practice Dr. Toni Winder, M.D. Dr. Randall Barley, Ph.D. Patient K.B. (Neurologist specializing (Experimental Surgery) (Recovering Stroke in ischemic stroke) Patient)
Policy and Practice Insert 1min video highlight of interviews
Background Cell Death Neurons
Background Microglia
Background Astrocytes
Background Reactive Astrocytes
Background
Background • Guo et al. (2013) - direct reprogramming of BRAAAINS reactive astrocytes to functional neurons using a single transcription factor, NeuroD1 BUT viral-mediated gene therapy in human patients is controversial!
Background Non-invasive Non-integrating Non-immunogenic Specific
Background • Alvarez-Erviti et al. (2011) – exosomes are an effective, non- invasive delivery mechanism for mRNA knockdown therapy • NJU China’s 2013 iGEM team • MIT’s 2013 iGEM team
Project Overview • Engineer microglia ( “ nomadocytes ” ) as non-immunogenic carriers of neural tissue-specific therapeutic genes Damaged Neurons Exosomes Microglia Reactive Astrocytes
Project Overview • Synthetic plasmid delivery system to target a neural reprogramming message ( NeuroD1 ) to reactive astrocytes Exosome Therapeutic Plasmid Plasmid Delivery System
Project Overview • This message will promote functional recovery after a TBI or stroke
Project Design Exosomal plasmid transport system: Exosome Target Sequence DNA-Binding Domain Lamp2B Rabies Virus Glycoprotein (RVG)
Project Design Nuclear targeting in recipient astrocyte: Exosome Nuclear Localization Signal (NLS) Protease Cut Site
Project Design Therapeutic plasmid: Exosome NeuroD1 RNA-OUT Sequence Astrocyte-Specific Promoter
Policy and Practice “A post -antibiotic era – in which common infections and minor injuries can kill – far from being an apocalyptic fantasy, is instead a very real possibility for the 21st Century.” – WHO, 2014 • Misuse of antibiotics • Potential for transfer of cassette to patient’s microbiome
Project Design Antibiotic free plasmid selection - RNA-IN/OUT • Native to E. coli Insertion Element 10 • Riboregulation of translation • Hybridization occludes ribosome binding site • Complementarity of RNA-OUT loop sequence and RNA-IN confers specificity RNA-IN RNA-OUT
Project Design 5’ 3’ A A U A G C G C C C
Project Design 5’ 3’ G C G A A C C U A C
Project Design Const. RNA-OUT Plasmid v pBAD ccdB Genome
Results Antibiotic free plasmid selection - RNA-IN/OUT: T4 Lysis Cassette pBAD pBAD-RNA-IN-Lysis RNA-OUT
Results Exosome Clover DNA-Binding Domain
Results
Results Predicted structure of RVG-Lamp2B-Clover exosomal protein:
Results Expression of RVG-Lamp2B-Clover exosomal protein in cell culture: A B C A. HEK control B. HEK + pcDNA3.0-Clover C. HEK + pcDNA3.0-RVG- D Lamp2B-Clover D. Murine microglia + pcDNA3.0-Lamp2B- Clover 20µm
Results Confirmed exosome production in HEK-293 cultures with transmission electron microscopy (TEM): 1 µm
Results Confirmed Clover fluorescence is localized to exosomes:
Results Developed the NeuroD1 therapeutic plasmid with the inclusion of IRES-Clover: Clover Doublecortin NeuN 20µm
Results: Summary ry • Established protocols for exosome isolation and purification • Successfully engineered an exosomal targeting system and demonstrated exosomal localization • Submitted RNA IN/OUT plasmid selection system, RVG-Lamp2B-Clover and TEV protease parts First steps towards a novel non-invasive cell-based gene therapy approach for human brains
Future Directions • Isolate primary astrocytes and microglia from mice rather than using immortalized lines • Validate exosomal packaging using animal model • Generate microglia from patient bone marrow cells
Policy and Practice • Collaboration with the Public Health Agency of Canada (PHAC): • Presented current and past iGEM projects • Demonstrated our commitment to biosafety • Feedback on current safety standards • Help update iGEM safety form
Policy and Practice Insert 30sec video highlight of interviews
Acknowledgements • The Public Health Agency of Canada (especially Kathrina Yambao & Kirsten Jacobsen) • Fan Mo, Ruzaan du Plooy, Doug Bray, and A. Will Smith (University of Lethbridge) • The Department of Chemistry and Biochemistry, the Canadian Centre for Behavioural Neuroscience, and the Kothe, Kovalchuk, McNaughton, Selinger, Gruber and Wieden labs at the University of Lethbridge.
Acknowledgements THANK YOU!
Thank You! Come check out our artwork in Hall C! It will be auctioned during our annual iGEM dinner and all proceeds will be going to charity ☺
References 1) Alvarez-Erviti, L., Seow, Y., Yin, H., Betts, C., Lakhal, S., & Wood, M.J.A. (2011). Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature Biotechnology, 29, 341-347. 2) Bi, F., Huang, C., Tong, J., Qiu, G., Huang, B., Wu, Q., Li, F., Xu, Z., Bowser, R., Xia, X-G., & Zhou, H. (2013). Reactive astrocytes secrete Icn2 to promote neuron death. PNAS, 110, 4069-4074. 3) Centers for Disease Control and Prevention. 1999. Traumatic brain injury in the United States: a report to Congress. Atlanta (GA): Department of Health and Human Services (US), CDC, National Center for Injury Prevention and Control. 4) Finkelstein, E.C., Corso, P.S., & Miller, T.R. 2006. The Incidence and Economic Burden of Injuries in the United States. New York: Oxford University Press; 5) Go, A.S., et al. 2014. Heart Disease and Stroke Statistics – 2014 Update: A Report from the American Heart Association. Circulation, 129, e28-e292. 6) Guo, Z., Zhang, L., Wu, Z., Chen, Y., Wang, F., & Chen, G. (2014). In vivo direct reprogramming of reactive glial cells into functional neurons after brain injury and in an Alzheimer’s disease model. Cell Stem Cell, 14, 188-202. 7) Heidenreich, P.A., et al. (2011). Forecasting the future of cardiovascular disease in the United States: a policy statement from the American Heart Association. Circulation, 123, 933-944. 8) Hinze, A. & Stolzing, A. (2012). Microglia differentiation using a culture system for the expansion of mice non-adherent bone marrow stem cells. Journal of Inflammation, 9, 12. 9) Kelley, L.A. & Sternberg, M.J.E. (2009). Protein structure prediction on the Web: a case study using the Phyre server. Nature Protocols , 4, 363 – 371. 10) MIT iGEM Team. (2013). Exosome mediated mammalian cell-cell communication. Retrieved from http://2013.igem.org/Team:MIT 11) Northern Brain Injury Association. (2014). Brain Injury Statistics. Retrieved from http://nbia.ca/brain-injury-statistics/ 12) NJU China iGEM Team. (2013). Biomissile. Retrieved from http://2013.igem.org/Team:NJU_China 13) Schjørring, S. & Krogfelt, K.A. (2011). Assessment of Bacterial Antibiotic Resistance Transfer in the Gut. International Journal of Microbiology, 2011: 312956. 14) United States Food and Drug Administration. (1998). Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy. Retrieved from http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/ CellularandGeneTherapy/ucm072987.htm 15) World Health Organization. (2014). Microbial resistance: global report on surveillance. Retrieved from http://www.who.int/ drugresistance/documents/surveillancereport/en/
Project Design No Arabinose pBAD ccdB No transcription Cell Survival
Project Design Arabinose Unsuccessful Transformation pBAD ccdB ccdB
Project Design Arabinose Successful Transformation pBAD ccdB ccdB ccdB
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