Engineering Microglia for Neural Cell Therapy LETHBRIDGE BRAINIACS: - - PowerPoint PPT Presentation

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Engineering Microglia for Neural Cell Therapy LETHBRIDGE BRAINIACS: - - PowerPoint PPT Presentation

Feb 09, 2023 220 likes •672 views

Engineering Microglia for Neural Cell Therapy LETHBRIDGE BRAINIACS: Dennis Bettenson Rhys Hakstol Harland Brandon Suneet Kharey Evan Caton Kelsey OBrien Rachael Chan Dustin Smith Billy Cowitz Zak Stinson Aubrey Demchuk Scott Wong

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Engineering Microglia for Neural Cell Therapy

Dennis Bettenson Harland Brandon Evan Caton Rachael Chan Billy Cowitz Aubrey Demchuk Graeme Glaister

LETHBRIDGE BRAINIACS:

Rhys Hakstol Suneet Kharey Kelsey O’Brien Dustin Smith Zak Stinson Scott Wong Hans-Joachim Wieden

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Background How many of you know someone who has been affected by:

Stroke Traumatic Brain Injury (TBI)

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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

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Policy and Practice

  • Dr. Toni Winder, M.D.

(Neurologist specializing in ischemic stroke)

  • Dr. Randall Barley, Ph.D.

(Experimental Surgery) Patient K.B. (Recovering Stroke Patient)

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Policy and Practice

Insert 1min video highlight of interviews

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Background

Neurons Cell Death

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Background

Microglia

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Background

Astrocytes

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SLIDE 9

Background

Reactive Astrocytes

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Background

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Background

  • Guo et al. (2013) - direct reprogramming of

reactive astrocytes to functional neurons using a single transcription factor, NeuroD1

BUT viral-mediated gene therapy in human patients is controversial!

BRAAAINS

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Background

Non-invasive Non-immunogenic Specific Non-integrating

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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
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Project Overview

  • Engineer microglia (“nomadocytes”) as non-immunogenic carriers
  • f neural tissue-specific therapeutic genes

Reactive Astrocytes Microglia Exosomes Damaged Neurons

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Project Overview

  • Synthetic plasmid delivery system to target a neural

reprogramming message (NeuroD1) to reactive astrocytes

Exosome Plasmid Delivery System Therapeutic Plasmid

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Project Overview

  • This message will promote functional recovery after a TBI or

stroke

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Project Design

Exosomal plasmid transport system:

Lamp2B Exosome DNA-Binding Domain Rabies Virus Glycoprotein (RVG) Target Sequence

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Project Design

Nuclear targeting in recipient astrocyte:

Nuclear Localization Signal (NLS) Exosome Protease Cut Site

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Project Design

Therapeutic plasmid:

Exosome Astrocyte-Specific Promoter NeuroD1 RNA-OUT Sequence

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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

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Project Design

Antibiotic free plasmid selection - RNA-IN/OUT

RNA-IN RNA-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

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Project Design

A A U A C C G G 3’ 5’ C C

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Project Design

A A U A C C G G 3’ 5’ C C

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Project Design

Plasmid Genome v RNA-OUT Const.

ccdB pBAD

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Results

Antibiotic free plasmid selection - RNA-IN/OUT:

RNA-OUT

pBAD

T4 Lysis Cassette

pBAD-RNA-IN-Lysis

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Results

Exosome DNA-Binding Domain Clover

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Results

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Results

Predicted structure of RVG-Lamp2B-Clover exosomal protein:

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Results

Expression of RVG-Lamp2B-Clover exosomal protein in cell culture:

A B C D

20µm

  • A. HEK control
  • B. HEK + pcDNA3.0-Clover
  • C. HEK + pcDNA3.0-RVG-

Lamp2B-Clover

  • D. Murine microglia +

pcDNA3.0-Lamp2B- Clover

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Results

Confirmed exosome production in HEK-293 cultures with transmission electron microscopy (TEM):

1 µm

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Results

Confirmed Clover fluorescence is localized to exosomes:

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Results

Developed the NeuroD1 therapeutic plasmid with the inclusion of IRES-Clover:

20µm

Clover Doublecortin NeuN

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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

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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
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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
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Policy and Practice

Insert 30sec video highlight of interviews

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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.

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Acknowledgements

THANK YOU!

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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 ☺

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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/

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Project Design

No Arabinose No transcription Cell Survival

ccdB pBAD

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Project Design

ccdB pBAD ccdB

Arabinose Unsuccessful Transformation

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Project Design

ccdB pBAD ccdB ccdB

Arabinose Successful Transformation