Combined Gene Therapy for GliomasThe linamarase/linamarin/glucose oxidase system as a potential

treatment for glioblastoma in the nude rat brain

Willie Girald-Rosa, PhDAutonomous University of Madrid, SpainMolecular Biology Center Severo Ochoa

Brain Tumors Primary brain tumors

◦ Glioblastoma multiform (GBM) Life expectancy: on average 14 months

Secondary brain tumors Classification of neoplasms affecting

the central nervous system:

WHO

Grade I Grade II Grade III Grade IV(GBM)

Low-grade High-grade

Not anaplasticwell-differentiated Anaplastic

Undifferentiated

Cancer stem cells cause tumour regrowth after apparently successful therapy

Initial GBM Minimal residual Recurrent GBM

Heterogeneous tumorResidual brain

tumor stem cells

Heterogeneous tumor regrowth from remaining tumor stem cells

Surgery, radio & chemo

Time

CSCs are proposed to persist in tumors as a distinct population and cause relapse by given rise

to new tumors

Glioma Cancer Stem cells (GCSCs) Possess characteristics associated with normal neural stem cells GCSCs are tumorigenic Specific Cellular Surface Markers

◦ Glioma Cancer Stem Cells (GCSCs): CD133+ & Nestin

Cancer CellsGCSCs

Glioma Cancer Stem Cell

Tumor recurrence

Conventional therapy targeting

tumor bulk

Tumor RegressionExisting GCSC replicate and differenciate

GCSCs targeted therapy

Eradication of GCSCs

Non-Conventional therapy targeting tumor bulk

Tumor eradication

Casava Plant, Cyanogenesis & lis-gene

Cyanohydrin group

Cyanogenic compound

LinamarinPlant Defense

Manihot esculenta

Epo-linamarase gene IRES pac

pILE 6.9Kb

amp r pBS Ori

pCM

V

U87MG-lis Cell line & CSCs(-5 & -7) lines

GBM tumor

Dissociation

Culturing with mitogens

Flow cytometry CD133+ CellsProliferation

Dissociation

Culturing with mitogens

Establishment of cell lines GCSCs-5 & GCSCs-7

Proliferation

-Removal of Growth factors-Addition of RADifferentiation

Neurophere

U87MGCells

pILE

puromacin

U87MG-lis

Actin

epo-lis-gene

Linamarase expression

Neural stem cells expression markers in neuropheres

To-Pro 3 Nestin

Mix

CD133

Multipotenciality analysis of the CSCs-5

β-III-Tubuline GFAP MixContrast

NG-2 MAP2/GFAP

Tumor generated by orthotopic injection of CSCs-5 cells in immunodeficent rat brain

Stereotaxic surgery

Rat’s brain with glioma

MRI

The linamarase/linamarin/glucose oxidase system

Lis/linReaction

GO/glucose Reaction

Aspergillus niger

The effect of the lis/lin/GO system

Cellular Death is accompained by mitochondrial fission and ATP depletion (Gargini R et.al. 2008).

Cellular Viability Assay to study the effect of lis/lin/GO system in the U87MG-lis in vitro

500 µg/ml linamarin5.5 mEU/ml GO48h @ 37ºC

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; a yellow tetrazole), is reduced to purple formazan in living cells.

Adenovirus type-5 and the E1-deficient replication-deficient adenoviral vector with

the epo-lis gene

ITR ITR0 4020 8060 100mu

E1 E2

E3

E4

Early Genes “E”

L1

L4L2

L5L3

Late Transcription

ITR CMVPromoter

epo Linamarase geneAn

Crucell Company, Holland

Linamarase expression in CSCs from patients

Immunofluorescent detection

Cellular Viability Assay to study the effect of lis/lin/GO system in the

GCSCs in vitro

Induction of Autophagy is associated to lis/lin/GO

•3-methyl Adenine: autophagy inhibitor•ShATG5: RNAi against gene ATG5

• Required for autophagy. Conjugates to ATG12 and associates with isolation membrane to form cup-shaped isolation membrane and autophagosome

Experimental Design

Stereotaxic surgery

Tumor confirmed by MRI

Combined Gene Therapy lis/lin/GO

U87MG-lis implantation

Local & Systemic Treatments

Delivery Systems

Osmotic Pump(OP)Model 2ML1Total Volume 2 ml10 µl/h/7days

Osmotic Pump(OP)Model 2001Total Volume 200 μl10 µl/h/7days

Intratumoral injectionHamilton 10ul 1µl/min

OP

Flow moderator

Catheter

Brain infusion cannula

Strategies to validate the lis/lin/GO system in the nude rat brain

Summary of Results Group II

A: before treatmentB: after treatment Kaplan Meier (p=0,291)

Death after a mean of 14 ± 1.5 days

Local TreatmentLis-Lin-GOx

Group IIIntracranial injections

250 mg/ml lin + 150 EU/ml GO

Death by toxicity

Death by Tumor

Survived

Summary of Results Group V-CRegression of the tumor after treatment

Beforetreatment

14 days post-treatment

MRI T123 days

post-treatment

MRI T223 days

post-treatment

MRI T145 days

post-treatment

Cocktail in OP:400 mg/ml lin20 EU/ml GODelivery rate:~2.25 µl/h

Linamarase & human MHCI detections in vivo in rat’s brain samples after treatment

Short-term toxic effect

M: markerR: rat treated local & systemically for 2 days 0-96 hours: rats treated local & systemically for different time pointsC1R: human lymphoid cells as + control

Conclusions

The killing effect of the lis/lin/GO system against CSCs and U87MG cell are very effective in vitro.

The lis/lin/GO system eliminates small glioma tumors in the brain of nude rats but in the majority of cases is toxic to the animal. The putative therapeutic window must be very narrow.

Systemic treatments, in our conditions, are not therapeutic probably due to the blood-brain barrier.