Hypoxia-targeted Gene Therapy of Tumors using Virus-directed Enzyme-Prodrug Systems Jeff Voegele December 4, 2012 (

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 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 1 o = 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.

Fold Induction = the ratio of transgene expression of hypoxic conditions relative to normoxic conditions. Graph (a) shows constructs containing the mCMV promoter Graph (b) shows constructs containing the mIL2 promoter

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 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 hypoxia- specific effect than HT1080 cells, so they were chosen for in vivo testing.

Comparison of HSVtk and NTR Prodrug- Activating Enzyme Systems in Human Primary Ovarian Cancer Cells Ad-VEGFmCMV-HSVtk and Ad-VEGFmCMV- NTR 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-VEGFmCMV- NTR 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 prodrug- activating enzyme transgenes to target hypoxic areas of tumors. VEGFmCMV was determined to be the strongest HRE- promoter 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 hypoxia- targeted gene therapy using HSV thymidine kinase and bacterial nitroreductase prodrug-activating genes in vitro and in vivo.” Cancer Gene Therapy (2011). 18, ; doi: /cgt ; published online 12 August Brown, JM. “Exploiting the hypoxic cancer cell: mechanisms and therapeutic strategies. Mol Med Today 2000; 6,