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Targeting and killing of malignant gliomas

by specific stem cells expressing a suicide gene

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

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

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

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

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

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

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)

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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 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.

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