1. { Targeting and killing of malignant gliomas by specific stem cells expressing a suicide gene

2.  Gliomas are tumors that arise from glial cells  Located in the brain or spinal cord  Current treatment: Neurosurgery, Radio- & Chemotherapy  After diagnosis, 30% of human patients live to 1 year and 14% live to 2 years.  Glioma cells can migrate and form new tumors Malignant Gliomas

3.  Have the ability to migrate to a pathological focus (e.g. to brain tumors)  Limitations: Cost, low population doublings, ability to subculture, and pathological focus Adult Stem Cells

4.  Different types of stem cells were tested and selected for, based on:  Ability to be subcultured  Highly proliferative  No antigenic surface markers  Ability to migrate to gliomas and track tumorous cells  Bone Marrow-derived Tumor-Infiltrating Cells (BM-TICs)  showed the most promise in these experiments Selecting the best cellular vehicle

5.  Transduced with a retro-viral vector containing the Thymidine Kinase gene from HSV & the green fluorescent protein gene.  Tumor infiltration and ability to track migrating tumor cells  Examined by Fluorescence Microscopy BM-TIC genetic modification

6. Gap junction formation?  Glioma cells dyed with DiI  BM-TICs are dyed with Calcein-AM  Intially two distinct populations after mixing (a)  Incubated for 3 hours  Glioma cells taken in Calcein-AM & BM-TICs have lost intensity  DiI is a membrane bound protein  Increase in Glioma cells Calcein-AM concentration  explained by Gap Junction communication

7. Ganciclovir & Gap Junctions  Rats with BM-TIC-tk-GFP injections were treated with Ganciclovir  Ganciclovir is a prodrug that reacts with viral thymidine kinase  Results in production of a toxic triphosphate  Incorporates into DNA and stops replication, signaling apoptosis  Gap Junctions between the tumor cells and therapeutic cells allow viral thymidine kinase diffusion  Rendering tumor cells susceptible to Ganciclovir

8. In vivo testing  Rats were injected with BM-TIC-tk-GFP, into or near tumors  4 days after injection the rats were treated with Ganciclovir daily for 10 days  Over 65% of treated rats showed long term survival  Statistical significance (P<0.001) % Surviving Animals

9. Future Treatment in humans  This method was highly effective in rats and shows promise for the treatment of malignant gliomas in humans  Thymidine kinase based gene therapy techniques have reached clinical trials References: Rachet B, Mitry E, Quinn MJ, et alRachet B, Mitry E, Quinn MJ, et al; Survival from brain tumours in England and Wales up to 2001. Br J Cancer. 2008 Sep 23;99 Suppl 1:S98-101 Goodenberger ML, Jenkins RB (2012). “Genetics of adult glioma”. Cancer genet. doi:10.1016/j.cancergen.2012.10.009 Miletic, Hrvoie, et. al. “Bystander Killing Of Malignant Glioma By Bone Marrow-Derived Tumor-Infiltrating Progenitor Cells Expressing A Suicide Gene.” Molecular Therapy: The Journal Of The American Society Of Gene Therapy 15.7 (2007): 1373- 1381.

10. Questions?