260 the effect of e1b 19kda adenoviral protein in combination with radiation therapy

260 The Effect of E1B 19kDa Adenoviral Protein in Combination with Radiation Therapy Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S102 CANCER TA[.]

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

S102

CANCER - TARGETED GENE THERAPY I

at a time In CT26, CAR-defi cient murine colon cancer cell lines,

the expression level of transgenes, is dramatically increased when

we transfected adenoviruses harboring transgenes such as luciferase

or GFP in conjugated with NCC-K1 The effect of NCC-K1 is also

repeated in mouse subcutaneous tumor model using CT26 cells

like in cell lines Furthermore, we could fi nd the dramatic effect on

tumor growth inhibition in mouse subcutaneous tumor model when

we treated adenovirus harboring thymidine kinase as a suicide gene

therapy, implying the successful gene delivery by NCC-K1 At that

time, it is possible to monitor the localization of adenoviruses at

regular time intervals using MRI Our fi ndings suggest that, in the

future, NCC-K1 is very easy and worthy for successful viral gene

delivery and noninvasive in vivo MRI tracking of targeted gene

therapy simultaneously

258 Relationship of Genetic Change and

Effi cacy of Viral Therapy in the Treatment of

Gliomas

Takahito Yazaki,1,2 Ryuichi Kanai,2 Takeshi Kawase.2

1 Neurosurgery, International Univ of Health and Welfare, Tokyo,

Japan; 2 Neurosurgery, School of Medicine, Keio University, Tokyo,

Japan.

Although HSV-1 mutants possessing deletions in both copies

animal experiments and clinical trials, their therapeutic effi cacy

was also markedly reduced In order to overcome this situation, we

concentrated on the use of tumor-specifi c promoter to express ICP34.5

selectively in malignant glioma cells As a molecular marker for

malignant glioma, we focused on the neural RNA binding protein,

Musashi1 We created, via homologous recombination, a novel

HSV-1 vector termed KeM34.5, which expresses ICP34.5 under

the transcriptional control of Musashi1gene promoter (P/musashi1)

Cytotoxicity mediated by KeM34.5 was signifi cantly enhanced in

human glioma cell lines, resulting in about 2 log increase of viral

yield, compared to its parental vector G207 We studied in this

time, whether genetic change as 1p/19q loss in gliomas is correlated

with therapeutic effi cacy of KeM34.5 in vivo glioma model This

virus showed a much higher therapeutic effi cacy in the gliomas

with 1p/19q loss, while maintaining the genetically intact gliomas

These results suggest that the oncolytic HSV-1 expressing ICP34.5

under the transcriptional control of Musashi1 gene promoter can be

a promising therapeutic agent for the treatment of malignant glioma

with 1p/19q loss

259 The Effects of Radiation on Adenoviral

Transgene Expression in Cancer Cells

Petri Nokisalmi,1 Maria Rajecki,1 Laura Ahtiainen,1 Vincenzo

Cerullo,1 Mikko Tenhunen,2 Sari Pesonen,1 Akseli Hemminki.1

1 Cancer Gene Therapy Group, Molecular Cancer Biology

Program & Transplantation Laboratory & HUSLAB & Finnish

Institute for Molecular Medicine, University of Helsinki and

Helsinki University Central Hospital, Helsinki, Finland;

2 Department of Oncology, Helsinki University Central Hospital,

Helsinki, Finland.

Genetically modifi ed adenoviruses have potential to kill cancer

cells by oncolysis due to viral infection per se, by anti-cancer

transgene expression and by activation of immunological responses

Adenoviruses have been safe in clinical cancer trials However, gene

therapy with adenoviruses alone is often not effective enough to

eradicate advanced tumors A common clinical practice is to combine

several treatment modalities in order to reach improved outcomes

Radiotherapy provides accurate and precise modes to treat different

cancer types and is widely combined with surgery and chemotherapy

Combination of cancer gene therapy with adenoviruses and

radiotherapy could lead to increased tumor control perhaps without

an increase in toxicity due to a non-overlapping side effect profi le The knowledge of interactions and potential synergy mechanisms is important when adenoviruses are combined to traditional treatments

and treatment protocols are optimized Methods and results: We

studied the effect of radiation on fi ve (Ad5cmv-luc, Ad5mdr-luc, Ad5ala-luc, Ad5cox2-luc and Ad5vegf-luc) promoters in PC-3MM2 prostate cancer cells We show that 8 Gy radiation dose increased transgene expression with all promoters Furthermore, FACS analysis

of prostate cancer cells infected with green fl uorescence protein (GFP) expressing Ad5GL and Ad5pk7GL viruses showed up to 8-fold increase in GFP expression To study the effect of radiation treatment on cellular proteins, we analyzed GFP expression also in two cancer cell lines stably expressing GFP: M4A4-LM3 breast cancer and LNM35-eGFP lung cancer cell line Cellular GFP expression was increased in both cell lines in a same manner as expression of transgenic GFP Moreover, radiation increased total mRNA levels up

to 2.5-fold in M4A4-LM3-cells These results imply that radiation generally activates cancer cells to produce more protein including viral proteins Finally we studied the effect of different radiation doses on luciferase expression in M4A4-LM3-cells infected with Ad5luc 0.5

Gy dose increased the amount of luciferase 1.6-fold and highest

2.4-fold increase was achieved with 12 Gy dose Conclusions: Radiation

increased adenoviral transgene expression regardless of transgene, transgene promoter or radiation dose Radiation also increased total mRNA-levels and cellular proteins in cancer cells These results imply that radiation can be utilized to increase the potency of adenoviral gene therapy due to general activation of gene expression/protein production, including therapeutic viral proteins

260 The Effect of E1B 19kDa Adenoviral Protein

in Combination with Radiation Therapy

Pyung-Hwan Kim,1 Jaesung Kim,2 Ji Young Yoo,1 A-Rum Yoon,2 In-Wook Kim,1 Hye Jin Choi,1,3 Jin Sil Seong,4 Chae-Ok Yun,1,2 Joo-Hang Kim.1,3

1 Graduate Program for Nanomedical Science, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea; 2 Brain Korea 21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic

of Korea; 3 Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea; 4 Department of Radiation Oncology, Yonsei University College of Medicine, Seoul, Republic of Korea.

Radiation therapy is a mainstay for anti-tumor therapeutic regimen for a variety of tumor types It triggers tumor cell apoptotic pathway via eliciting direct DNA damage or indirectly by inducing the formation of oxygen radicals In efforts to augment the anti-tumor effi cacy of oncolytic adenovirus (Ad), we have examined the effect of E1B 19kDa, a strong anti-apoptotic protein present in oncolytic Ad, in combination with radiation therapy An enhanced cancer cell killing

combination with radiotherapy than single treatment alone Notably, the highest cytotoxicity was observed in cancer cells treated with Ad-DE1B19/55 and irradiation Signifi cant induction of apoptosis was noted in cancer cells treated with Ad-∆E1B19/55 and irradiation

as demonstrated by fl ow cytometry and TUNEL assays Moreover, enhanced levels of p53, phospho-p53, phospho-Chk1, phospho-Chk2, PI3K, phospho-AKT and cleavage of PARP and caspase-3 were observed when cells were treated with Ad-∆E1B19/55 plus radiation,

play a critical role in blocking the effect of irradiation-induced

apoptosis Signifi cant therapeutic benefi t also translated in vivo when

oncolytic Ads and radiation were combined Moreover, tumors treated

Molecular Therapy Volume 17, Supplement 1, May 2009

CANCER - TARGETED GENE THERAPY I

with Ad-∆E1B19/55 plus radiation showed large areas of necrosis and

abundant apoptosis with induction of p53 In relative comparison,

combination of Ad-∆E1B19/55 and radiation was superior over that of

Ad-∆E1B55 and radiation combination treatment Overall, this study

presents strong therapeutic rationale for combination of radiation

therapy and E1B 19kDa-deleted oncolytic Ad

261 Adenovirus Infection of Epithelial Cancer

Cells

Robert Strauss,1 Pavel Sova,1 Ying Liu,1 ZongYi Li,1 Sari Pesonen,2

Akseli Hemminki,2 Pascal Fender,3 Andre Lieber.1

1 Medicine, University of Washington, Seattle, WA; 2 Cancer Gene

Therapy Group, University of Helsinki, Helsinki, Finland; 3 Institut

de Biologie Structural, Grenoble, France.

The majority of solid cancers are of epithelial origin A hallmark of

epithelial cells are tight and adherens junctions Tight and adherens

junctions seal intercellular spaces and signifi cantly limit the perfusion

of anti-tumor agents such as drugs, antibodies, and immune cells

therapeutic modalities The epithelial phenotype of cancer cells also

represents a barrier to infection with commonly used adenoviruses

that target CD46 or the coxsackie-adenovirus receptor (CAR), due

to trapping of these receptors in tight and adherens junctions which

explains, in part, why these serotypes were ineffi cient in cancer

gene therapy We found however, that specifi c human species B

adenoviruses, namely Ad3, Ad7, Ad11, and Ad14 (AdB-group 2)

that use a yet unknown receptor (receptor X), which is different from

CAR and CD46, effi ciently infect epithelial cancer cells This makes

these serotypes potential tools for virotherapy of cancer as well as for

gene transfer into normal epithelial tissue In addition, these serotypes

are important pathogens, exemplifi ed by recent outbreaks of a highly

pathogenic new Ad14 stain So far, our studies on mechanisms of

AdB-group 2 infection of epithelial cells have shown: i) These

Ads use at least two binding moieties on the cell membrane The

C-terminal part of the Ad fi ber (the fi ber knob) binds to sulfated

carbohydrate chains of heparin-sulfate proteoglycans (HSPG) This

Ad3knob – interaction with heparin sulfate glucosaminoglycans

(HS-GAGs) allows for subsequent high affi nity attachment and/or access

to receptor X, whereby receptor X is either the protein part of the

HSPG that interacts with the Ad3 knob or an independent non-HSPG

protein ii) Ad binding to epithelial cells involves both the Ad3 fi ber

and the Ad3 penton or a composite fi ber-penton moeity formed in

Ad3 virions iii) During virus replication, Ad pentons and fi bers

self-assemble in dodecahedra (PtDd) formed through interaction of 12

penton bases with protruding fi bers These PtDd are thought to disturb

tight junctions, thus favoring lateral virus spreading iv) Recombinant

PtDd (produced in insect cells) can enter epithelial cancer cells and

can faciliate uptake of Ads (and potentially other tumor agents) for

which tight and adherens junctions represent a barrier

262 Serotype Chimeric and Fiber Mutated Adenovirus Ad5/19p-HIT for Targeting Renal Cancer and Untargeting the Liver

Iulia Diaconu,1,2 Laura Denby,3 Sari Pesonen,1,2 Vincenzo Cerullo,1,2 Gerd J Bauerschmitz,4 Kilian Guse,1,2 Maria Rajecki,1,2 João D Dias,1,2 Kimmo Taari,5 Anna Kanerva,1,2,6 Andrew H Baker,3 Akseli Hemminki.1,2

1 Cancer Gene Therapy Group, Molecular Cancer Biology Program & Transplantation Laboratory & Haartman Institute &

Finnish Institute for Molecular Medicine, University of Helsinki, Helsinki, Finland; 2 HUSLAB, Helsinki University Central Hospital (HUCH), Helsinki, Finland; 3 British Heart Foundation Glasgow Cardiovascular Research Center, University of Glasgow, Glasgow, United Kingdom; 4 Department of Obstetrics and Gynecology, Heinrich-Heine University, Düsseldorf, Germany; 5 Department of Urology, Helsinki University Central Hospital (HUCH), Helsinki, Finland; 6 Department of Obstetrics and Gynecology, Helsinki University Central Hospital (HUCH), Helsinki, Finland.

Despite some recent advances, patients with advanced renal cell carcinoma (RCC) cannot usually be cured Alteration of the natural tropism of adenoviruses may permit more specifi c gene transfer to target tissues The aim of this study was to utilize novel targeting moieties for adenoviral gene therapy of RCC Previous work in rats suggested that utilization of Ad5/19p (Ad5 capsid with Ad19p fi ber) with kidney vascular targeting moieties HTTHREP (HTT), HITSLLS (HIT) and APASLYN (APA) placed into the fi ber knob might be useful for targeting kidney vasculature Therefore, we sought to investigate the utility of Ad5/19p variants for gene delivery to human RCC cell lines, clinical samples and orthotopic murine models of metastatic RCC Six different human RCC cell lines were infected with Ad5/19p variants but only Ad5/19p-HIT showed increased transduction, and only in one cell line Thus, we analyzed human normal and cancerous kidney specimens fresh from patients, which might better mimic the three dimensional architecture of clinical tumors and found that Ad5/19p-HIT showed transduction levels similar to Ad5 In mice, we found that intraperitoneal and intravenous Ad5/19p-HIT transduced tumors at levels comparable to Ad5, while intratumoral Ad5/19p-HIT was even superior to Ad5 Liver tropism was signifi cantly reduced

in comparison to Ad5 Improvements in tumor to liver transduction ratios suggested that Ad5/19p-HIT may be promising for systemic gene delivery to kidney tumors

263 An Effi cient Targeted Gene Therapy Using Brain Tumor-Specifi c Promoter

Toshio Yawata,1 Eri Ishida,1 Yu Kawanishi,1 Masakazu Tamura,1 Keiji Shimizu.1

1 Neurosurgery, Kochi Medical School, Nankoku, Kochi, Japan.

Despite many efforts to develop effective therapy, the outcome

of malignant glioma remains poor Gene therapy for this disease using retroviral vector is attractive, because the virus can infect only mitotic cells Previously, we reported the eradication of mouse glioma

by retroviral-mediated gene therapy In this study, a tumor-specifi c targeting system was studied to develop the effective and safe gene therapy We searched for genes expressing at high frequency in brain tumors but not in normal human astrocyte (NHA) among cancer testis antigen (CTA) genes MAGEA3 and SSX4 were identifi ed as tumor-specifi c genes The promoter of both genes was cloned into luciferase reporter vector and the activity was measured in glioma, telomerase-immortalized fi broblast and normal human astrocyte cells The SSX4 promoter but not MAGEA3 showed the tumor-specifi c activity The minimal promoter of SSX4 was defi ned as a

256 bp fragment upstream of transcriptional start site In order to defi ne a useful tumor-specifi c promoter for targeting in context of retroviral-mediated gene therapy, the SSX4 promoter were used to