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Hypoxia-targeted Gene Therapy of
Tumors using Virus-directed
Enzyme-Prodrug Systems
Jeff Voegele
December 4, 2012
(http://www.markergene.com/WebNewsletter10.5.htm)
Solid Tumors
• Make up more than 90% of all human cancers.
• Form from a single mutated cell, which then spreads to
surrounding tissue.
• A tumor must obtain its own blood supply to grow, and
it does this by stimulating the growth of surrounding
blood vessels to feed the tumor (angiogenesis).
– Tumor blood vessels are typically highly irregular, which
decreases the efficiency of oxygen delivery to the cancer
cells.
– Tumor cells that are deprived of oxygen are known as
hypoxic cells.
Hypoxia
• Most solid tumors have hypoxic regions which
are more resistant to radiotherapy and
chemotherapy as opposed to well-oxygenated
(normoxic) regions
• Related to malignant progression, increased
invasion, angiogenesis (growth of new blood
vessels), and increased risk of metastasis.
HIF-1
• Hypoxia-inducible factor 1
• Overexpression of α-subunit is thought to lead to
increased tumor aggression
• In hypoxic regions, HIF-1 activates transcription
by binding to hypoxic-response elements (HREs)
within promoter regions, leading to
overexpression of specific proteins in the tumor.
– Vascular Endothelial Growth Factor (VEGF)
– Erythropoietin (EPO)
Significance of HREs
• The HREs of VEGF and EPO have been shown
to be sensitive to hypoxic conditions and have
been used in many gene therapy studies to
target hypoxia.
• These HREs thus give us the ability to
selectively target hypoxic areas of a tumor for
therapeutic gene expression.
Aim of this Research
• To develop antitumor therapies that target
hypoxic regions.
• Combine this therapy with traditional cancer
therapy to kill normoxic and hypoxic regions.
Virus-directed Enzyme Prodrug
Therapy (VDEPT)
• The use of a virus as a vector is a well-established
method to deliver a target gene to a tissue for
therapy.
• A gene encoding a prodrug-activating (“suicide”)
enzyme is first delivered to the tissue by a viral
vector.
• The suicide enzyme then metabolizes a non-toxic
prodrug into a toxic compound.
• The toxic compounds diffuse to and kill
neighboring cells via the “bystander effect.”
Prodrug-activating Genes
• Herpes Simplex Virus Thymidine Kinase
(HSVtk)
• Ganciclovir (GCV) is its prodrug
• Bacterial Nitroreductase (NTR)
• CB1954 is its prodrug
TJ Harvey et al. (2011)
• Constructed plasmid and adenoviral vectors
encoding HSVtk and NTR suicide genes, under
control of either VEGF or EPO HREs combined
with either the minimal cytomegalovirus (mCMV)
or minimal interleukin-2 (mIL-2) promoter.
• Compared cytotoxic effects of these constructs in
established cancer cell lines and in primary
human tumor cell cultures in vitro.
• In preparation for clinical trials, they examined
the power of the optimal adenoviral vectors in
human tumor xenograft models in mice in vivo.
Human Cell Line Cultures
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UMUC3 = Urothelial Carcinoma Cell Line
SKOV3 = Ovarian Carcinoma Cell Line
OVCA433 = Ovarian Carcinoma Cell Line
HCT116 = Human Colon Cancer Cell Line
JON = Bladder Carcinoma Cell Line
HT1080 = Human Fibrosarcoma Cell Line
Patient-Derived Tumor Specimen
Culture
• Ovarian 1o = Primary Ovarian Cancer Cells
(derived from ascitic fluids of patients)
Western Blot Analysis of HIF-1α
Expression
• Each cancer cell line was subjected to
normoxic (N) and hypoxic (H) conditions for 17
hours.
• HIF-1α expression was monitored by a
western blot.
Comparison of VEGF and EPO HREs
• The cancer cell lines were transfected with
luciferase reporter plasmids, which did or did
not contain 5 repeats of VEGF or EPO HREs.
• The HREs were inserted upstream from either
an mCMV or an mIL-2 minimal promoter.
Graph (a) shows constructs
containing the mCMV promoter
Graph (b) shows constructs
containing the mIL2 promoter
Fold Induction = the ratio of
transgene expression of hypoxic
conditions relative to normoxic
conditions.
• Optimal hypoxia-inducible HRE-promoter
system identified as VGEFmCMV.
• HCT116 and HT1080 cells used from this point
on.
In vitro Comparison of HSVtk and NTR
Prodrug-Activating Enzyme Systems
• A panel of recombinant replication-defective
adenoviral vectors that contained either HSVtk or NTR
therapeutic transgenes was created.
• These vectors were used to compare the cytotoxicity of
the two therapeutic transgenes (HSVtk and NTR) under
normoxic and hypoxic conditions.
• Ad-CMV-HSVtk and Ad-CMV-NTR (using full CMV
promoter) vectors used as positive controls. These
target both normoxic and hypoxic cells.
• Compare cytotoxicity of hypoxia-targeting transgene
(Ad-VEGFmCMV-HSVtk/NTR) to positive controls.
Therapeutic Constructs
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Ad-CMV-NTR
Ad-CMV-HSVtk
Ad-mCMV-NTR
Ad-mCMV-HSVtk
Ad-VEGFmCMV-NTR
Ad-VEGFmCMV-HSVtk
Mock Trials
Positive Controls (full CMV promoter)
Hypoxia-targeted Constructs
In vitro Results
• The positive controls showed strong cytotoxic effects
under both normoxic and hypoxic conditions in each
cell line.
• The hypoxia-inducible transgenes showed cytotoxic
effects in the presence of their respective prodrugs,
but not in their absence, under hypoxic conditions.
• In all cases, the effectiveness of the hypoxia-inducible
transgenes in hypoxic conditions was similar to that of
the positive controls.
• HCT116 cells showed a more pronounced hypoxiaspecific effect than HT1080 cells, so they were chosen
for in vivo testing.
Comparison of HSVtk and NTR ProdrugActivating Enzyme Systems in Human
Primary Ovarian Cancer Cells
• Ad-VEGFmCMV-HSVtk and Ad-VEGFmCMVNTR viruses were introduced into primary
ovarian cancer cells to examine their
cytotoxicity under hypoxic conditions.
• Adenoviruses with the mCMV promoter and
the full CMV promoter were included as
negative and positive controls, respectively.
Negative control (Ad-mCMV-HSVtk)
shows no significant cytotoxic effect.
Positive control (Ad-CMV-HSVtk) shows
efficient cytotoxicity under both
normoxic and hypoxic conditions,
especially for the NTR/CB1954 system.
Both hypoxia-inducible transgenes show
significant cytotoxic effects under hypoxic
conditions, with the HSVtk/GCV system
resulting in about 65% cell death and the
NTR/CB1954 system resulting in about
97% cell death.
In vivo Testing of Hypoxia-Inducible
System
• Based on prior results, the Ad-VEGFmCMVNTR virus/CB1954 prodrug-activating system
was chosen for in vivo therapeutic testing.
• Ad-VEGFmCMV-NTR was injected
intratumorally into HCT116 xenografts
(transplanted tumors) in nude mice.
• Tumor volumes were recorded daily over a
period of 15 days as the CB1954 prodrug was
administered.
Ad-VEGFmCMV-NTR + CB1954
shows a significant delay in
tumor growth over the 15 day
period as opposed to the case
without the prodrug.
Ad-mCMV-NTR, administered
with or without CB1954, did
not show a significant effect
on tumor growth.
Ad-VEGFmCMV-NTR showed
significant reduction in tumor
growth compared to Ad-mCMV-NTR
groups only in the presence of
CB1954, and not in its absence.
Hypoxic Localization of Transgene
Expression
• Immunostaining experiments were performed
to test the hypothesis that the enhanced
cytotoxicity of the Ad-VEGFmCMV-NTR virus
seen in vivo is a consequence its ability to
selectively target hypoxic areas of the tumor.
• The tumors were removed after the mice
were killed and adjacent regions were stained.
• anti-Glut1 antibody to mark hypoxic areas
• anti-NTR antibody to mark adenoviral transgene
expression
Similar staining patterns seen between
Glut1 (showing hypoxic regions) and
NTR (showing adenoviral expression)
staining, revealing that viral expression was
localized to hypoxic regions of the tumor.
As expected, no NTR staining seen in the
absence of the virus (j).
Conclusions
• HREs of VEGF and EPO are capable of driving prodrugactivating enzyme transgenes to target hypoxic areas of
tumors.
• VEGFmCMV was determined to be the strongest HREpromoter system to direct hypoxia-specific transgene
expression.
• Both hypoxia-inducible transgenes (HSVtk and NTR) showed
significant cytotoxic effects under hypoxic conditions, but the
NTR transgene was determined to be more efficient.
• The NTR transgene showed significant reduction in tumor
volume in the presence of CB1954, and not in its absence.
• Based on immunostaining experiments, hypoxia-inducible
transgene expression appears to be localized only to hypoxic
areas within the tumor.
References
• TJ Harvey, IM Hennig, SD Shnyder, PA Cooper, N Ingram, GD
Hall, PJ Selby, and JD Chester. “Adenovirus-mediated hypoxiatargeted gene therapy using HSV thymidine kinase and
bacterial nitroreductase prodrug-activating genes in vitro and
in vivo.” Cancer Gene Therapy (2011). 18, 773-784; doi:
10.1038/cgt.2011.43; published online 12 August 2011.
• Brown, JM. “Exploiting the hypoxic cancer cell: mechanisms
and therapeutic strategies. Mol Med Today 2000; 6, 157-162.