839 the use of third generation oncolytic HSV 1 expressing luciferase for demonstration of real time biodistribution and dynamics

839 The Use of Third Generation Oncolytic HSV 1 Expressing Luciferase for Demonstration of Real Time Biodistribution and Dynamics Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The Am[.]

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S322

CANCER-ONCOLYTIC VIRUSES III

834 Incorporation of a Mutated E1A and a

Chimeric 5/3 Fiber Improved the Therapeutic

Ef cacy of a Novel CRAd (Ad-F512) on

Disseminated Ovarian Cancer

Veronica M Lopez,1 Angel A Rivera,2 Lorena Benedetti,1 Diego

L Viale,1 Kristopher J Kimball,3 Minghui Wang,2 Alicia J Bravo,4

Christine Dorvault,5 Joanne Douglas,2 Zeng B Zhu,2 Ronald D

Alvarez,3 David T Curiel,2 Osvaldo L Podhajcer.1

1 Laboratory of Molecular and Cellular Therapy, Instituto

Leloir-CONICET, Buenos Aires, Argentina; 2 Division of Human Gene

Therapy, University of Alabama, Birmingham, AL; 3 The Division

of Gynecologic Oncology, UAB, Birmingham, AL; 4 Hospital Eva

Peron, Buenos Aires, Argentina; 5 The Division of Pathology, UAB,

Birmingham, AL.

We have previously developed a new CRAd where E1A activity

is driven by a 0.5 kb fragment of the SPARC gene promoter (SPPr)

This novel CRAd (termed Ad-F512) was designed to target both

the malignant and the tumor-associated stromal compartment of the

tumor mass (Lopez et al, PlosOne 2009) In order to further improve

speci city and retargeting, we prepared different versions of the

CRAd where SPPr is driven mutated E1A variants Moreover, the

CRAd was pseudotyped with a chimeric Ad5/3(shaft/knob)  ber

Ovarian cancer is one of the leading gynecologic malignancies with no

effective treatment at advanced stages if conventional chemotherapy

fails In the present study we assessed the different CRAd variants

in ovarian cancer models

Initial studies were designed to establish the infective capacity

of the new vectors We observed in 3 out of 4 ovarian cancer cell

lines a 2-50 fold increase when Ad-SV40-luc 5/3 was compared to

SV40-luc 5/5 Moreover, F512-luc 5/3 was as active as

Ad-SV40-luc 5/3 Next, we constructed four CRAds where SPPr drove

the activity of wild type E1A, E1A deleted in the Rb binding motif,

in the p300 motif or both and measured the ef cacy in the 4 ovarian

cancer cell lines By using the MTS assay we observed that the better

lytic capacity was obtained with the CRAd containing the Rb deletion

(Ad-F512-E1∆Rb 5/3)

In order to establish the potential clinical utility of

Ad-F512-E1∆Rb 5/3 we also assayed its replication capacity in a most rigorous

preclinical system, which is slices obtained from fresh tissue explants

of human ovarian carcinomas compared to normal ovary

Ad-F512-E1∆Rb 5/3 replicated in three out of four human ovarian carcinomas

but showed no replication in 3 normal ovary samples It is important

to mention that Ad-wt 5/3 could replicate in both malignant and

normal samples Histochemical analysis for SPARC expression

levels, presence of stroma and virus replication was also performed

on the fresh explants

Finally, we examined the in vivo lytic capacity of Ad-F512-E1∆Rb

5/3 in a xenograft model of disseminated intraperitoneal ovarian

cancer by using a bioluminescent imaging follow up We found that

four i.p injections of 1010 v.p inhibited almost 50% of tumor growth,

(assessed using caliper, by signal intensity and by weight) in animals

treated with Ad-F512-E1A∆Rb 5/3 compared to the controls

In conclusion, Ad-F512-E1A∆Rb 5/3 demonstrated a strong killing

effect on ovarian cancer cells in vitro, and in vivo on disseminated

tumors as well as a high replication capacity in fresh human ovary

cancer explants Ad-F512-E1A∆Rb 5/3 appears safe since no

replication was observed in normal human ovary raising its potential

use in the clinics

835 Inhibition of Autophagy Has Negative Impact on Virus Production

Ilya V Ulasov,1 Atique U Ahmed,1 Bart Thaci,1 Natalya V

Kaverina,1 Maciej S Lesniak.1

1 Surgery, Brain Tumor Center, The University of Chicago, Chicago, IL.

Introduction Autophagy is a type of cell death where increased

numbers of autophagosomes appear in cytoplasm before cell death, without signs of necrosis or apoptosis In the literature, several therapeutic approaches that induce autophagy are described One of them is using oncolytic adenoviruses Preclinical studies conducted

on several types of tumor cell lines have revealed evidence that autophagy is associated with viral infection We are conducting this study to answer whether autophagy has an impact in virus replication

and toxicity Methods Chemical inhibition of virus associated

autophagy by 3-Ma (inhibitor of autophagosome maturation), BAF A1 (PI3 kinase inhibitor) and Rapamycin (mTOR inhibitor) The effect of blocking was evaluated based on: viral mediated toxicity (PI- staining); viral induced apoptosis (Caspase-3 cleaved study); viral

expression (E1A replication and progeny release) Results Inhibition

of autophagosome maturation led to decreased incorporation of acridine orange(AO) and LC3 downregulation along with decreased overall toxicity in human glioma cells regardless of p53 status

Real time PCR data obtained from N10, U87 and U118 cells lines demonstrated reduction of E1A expression in the group treated with S-pK7 following by 3Ma inhibition Further analysis of

CRAd-S-pK7 progeny released corroborated with real time PCR data and

showed 2-10 fold of reduction during 3-Ma inhibition Conclusions

and Future Directions Experiments performed on cell lines have

revealed that inhibition of autophagy with 3-Ma inhibitor blocks CRAd-S-pK7 replication and oncolysis Evidence suggests that the autophagy pathway has a great in uence in viral replication Impaired autophagy reduced the ef ciency of oncolytic virus application This study will broaden our knowledge regarding virus-induced cell death and will shed some new insight on possible strategies using oncolytic

adenovirus as part of combined therapy with drugs or radiation

836 Armed and Transcriptionally Driven Oncolytic HSV-1 Enhances Antitumor Ef cacy In

Vitro and In Vivo

Ji-Young Yoo,1 Amy Haseley,1 Anna Bratasz,2 Kimerly Powell,2

Balveen Kaur.1

1 Neurological Surgery, The Ohio State university, Columbus, OH; 2 Small Animal Imaging Center, The Ohio State University,

Columbus, OH.

Oncolytic viruses (OV) are an exciting biological therapy relying

on tumor speci c replication and lysis obyOV First generation attenuated oncolytic herpes simplex virus-1 (HSV-1) have been tested

in human patients, and found to be safe, but signi cant ef cacy has not yet been proven Transcriptional targeting of viruses wherein deleted viral genes in  rst generation OV are reinserted under the governance of tumor speci c promoters is a promising way to enhance their tumor speci c replication and ef cacy In this study,

we have generated a novel third generation OV (rQnestin34.5-Vstat120), within the backbone of HSV-1 deleted for ICP34.5 and

ICP6 viral genes rQnestin34.5-Vstat120, is a transcriptionally driven

OV, wherein ICP34.5 is reinserted in the doubly attenuated HSV-1 backbone under the control of glioma speci c nestin promoter, and expresses anti-angiogenic Vstat120 under an immediate early viral promoter We have previously shown potent antiangiogenic and antitumorigenic effects of Vstat120 in human glioma in vivo We have con rmed the functionality of ICP34.5 produced by

rQnestin34.5-Vstat120 in glioma cells with high nestin expression In high nestin expressing glioma cell lines/primary cells (U251, U87∆EGFR, and

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S323

CANCER-ONCOLYTIC VIRUSES III

X12-V2), rQnestin34.5-Vstat120 showed increased viral replication (25.8-fold (p=0.0065), 21-fold (p=0.0018), and 817-fold (p=0.0110) respectively), and increased glioma cytotoxicity (88.7% (p=0.0004), 90.6% (p=0.0001), and 76.8% (p=0.0014)) relative to HSVQ infected glioma cells In contrast little or no signi cant increase in virus replication and cytotoxicity was observed in glioma cells with low nestin expression (T98G and Gli36∆5), or in primary non transformed human endothelial cells (nestin negative) Collectively these results indicated nestin speci c increased viral replication and cytotoxicity

of rQnestin34.5-Vstat120

Further we have confirmed early and efficient secretion of Vstat120 in glioma cells infected with rQnestin34.5-Vstat120 The functionality of Vstat120 produced by rQnestin34.5-Vstat120 was veri ed by its ability to inhibit endothelial cell migtration and tube formation in vitro To test its antitumor ef cacy was mice with subcutaneous U251T3 glioma (150-250 mm3) were injected with PBS, rQnestin34.5 (transcriptionally driven OV), rQVstat120 (double attenuated expressing Vstat120 OV), or rQnestin34.5-Vstat120 (transcriptionally driven and Vstat120 expressing OV) Mice were evaluated for tumor growth A signi cant increase in anti-tumor effect was observed between rQnestin34.5-Vstat120, and rQnestin 34.5, (p=0.0198) and between rQnestin34.5-Vstat120, and rQVstat120 (p=

0.0429) We are now in the process of testing therapeutic ef cacy of rQnestin34.5-Vstat120 in intracranial tumor model MRI imaging of mice bearing intracranial tumors has revealed increased antitumor ef cacy of rQnestin34.5-Vstat120 against intracranial gliomas This study has led to the development of a novel transcriptionally retargeted and “armed” oncolytic virus

837 Determining Adenovirus Titers Following Extraction of the Virus from Tumors in the Presence of Anti-Adenovirus Antibodies

Debanjan Dhar,1 Karoly Toth,1 William S M Wold.1

1 Molecular Microbiology & Immunology, Saint Louis University,

St Louis, MO.

Oncolytic adenovirus (Ad) vectors are being investigated as possible therapies for human cancer These vectors are generally injected into the tumor, and multiple injections are usually necessary for the vector to be effective When the vector is injected into a nạve immunocompetent animal, an immune response is mounted against the vector Immune effector cells and antibodies (Ab) in ltrate the site

of injection Determining the Ad vector titer present inside the tumor

or any other tissue is challenging since the neutralizing Ab (NAb) could affect the results Even if the NAbs are physically separated from the infectious vector within the tumor microenvironment,

as soon as the tumor is disrupted in order to titer the vector, the NAbs could bind to the vector and neutralize its infectivity We have shown previously that Syrian hamsters are a good model to evaluate oncolytic Ad5-based vectors Normal tissues of hamsters are permissive for Ad5, and hamsters are immunocompetent In the current study, we used hamsters to determine the best way to determine the infectious vector titer from an immunocompetent animal and also from animals having preexisting immunity against the vector We also investigated whether the infectious vectors that are extracted from tumors and titered in a standard TCID-50 assay are intracellular within the tumor microenvironment or are extracellular trapped in the tumor architecture As control we used hamsters that were immunosuppressed before the vector was injected into the tumor and were kept immunosuppressed throughout the study Tumors were isolated at various time points, chopped into small pieces, and separated into three different tubes The tumor in  rst tube was homogenized by bead-beating, freeze thawed, and sonicated to release the intracellular vector, and then titered by TCID-50 assay The second tube was treated with collagenase-dispase to isolate single cells from the tumor The cells were washed and treated with Proteinase K to

get rid of the NAb, sonicated, and assayed for infectious vector Since high amounts of Proteinase K are detrimental to the cell, the optimal concentration of Proteinase K was determined in a separate experiment so that Proteinase K affected the extracellular NAb and not the tumor cell and the intracellular vector We found that most

of the vector recovered from the tumor is intracellular We also found that Collagenase-dispase along with Proteinase K treatment

or extensive washing is an effective method to eliminate NAb during vector extraction from tumors, allowing accurate measurement of infectious vector titers in a tumor

838 Abstract Withdrawn

839 The Use of Third Generation Oncolytic HSV-1 Expressing Luciferase for Demonstration of Real-Time Biodistribution and Dynamics

Yihan Wu,1 Yasushi Ino,2 Tomoki Todo.2

1 Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 2 Department of Neurosurgery and Translational Research Center, The University of Tokyo, Tokyo, Japan.

Background G47∆ is a third generation oncolytic herpes

simplex virus type 1 (HSV-1) containing deletions in the γ34.5 and α47 genes and an inactivating LacZ insertion in the UL39 (ICP6) gene Previous studies have shown that G47∆exhibits an ef cient oncolytic activity in a variety of tumor cells, yet minimal toxicity

in normal tissues In order to optimize the route of administration

and dosing for various types of cancer, it is essential to assess the in

vivo distribution and the time course of viral infection in relation to

ef cacy Conventionally, biodistribution studies required sacri cing and obtaining tissue samples from animals at different time points after virus administration In this study, we newly constructed a third generation oncolytic HSV-1 expressing luciferase, and used it to demonstrate a real time biodistribution of the virus in a non-invasive

fashion Methods

A third generation, armed oncolytic HSV-1 termed T-luc was constructed by inserting the CMV promoter-driven, luciferase gene into the G47∆backbone, using the bacterial arti cial chromosome-mediated, recombinant HSV-1 construction system (T-BAC system) After limiting dilutions three times, the  nal virus clone was selected

and the correct construction con rmed by Southern blot analyses In

vitro studies on cytopathic effect and viral replication ability were

performed using Vero and Neuro2a (murine neuroblastoma) cells

In vivo studies were performed using A/J mice bearing syngeneic

Neuro2a tumors or athymic mice bearing U87MG (human malignant

glioma) tumors The in vivo luciferase expression, re ecting the

fresh infection by T-luc, was measured at different time points after intratumoral or intravenous administration with T-luc using

the in vivo imaging system (IVIS, Xenogen) Results The in vitro

cytopathic effect and replication capability of T-luc were comparable

to those of G47∆ in both cell lines tested The in vivo antitumor

ef cacy of T-luc was also similar to that of G47∆ when tested in A/J mice bearing subcutaneous Neuro2a tumors When T-luc was injected intratumorally into subcutaneous Neuro2a tumors, the local luciferase expression was detected up to day 7 In athymic mice with subcutaneous U87MG tumors, intratumoral injections with T-luc resulted in local expression of luciferase for more than 14 days The T-luc infection was not detected from any other organs at any time point after an intratumoral administration Contrarily, an intravenous administration with T-luc in animals with subcutaneous tumors resulted in a high expression of luciferase in the liver and the tail (the injection site) as well as in tumors In contrast with the luciferase expression in tumors which gradually increased by day 7, the luciferase expression in the liver rapidly vanished by the next day of viral injection The luciferase expression in intracranial

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S324

CANCER-ONCOLYTIC VIRUSES III Neuro-2a tumors was detectable up to day 6 Conclusion The newly

constructed, luciferase-expressing HSV-1, T-luc, is a useful tool for

studying biodistribution and dynamics of oncolytic HSV-1

840 Highly Attenuated Vaccinia Virus as a

Potential Oncolytic Agent for Cancer Virotherapy

Takafumi Nakamura,1,2 Minoru Kidokoro,3 Mina Hikichi,1

Hisatoshi Shida,4 Hideaki Tahara.1

1 Institute of Medical Science, Tokyo University, Tokyo,

Japan; 2 PRESTO, Japan Science and Technology Agency,

Kawaguchi, Japan; 3 National Institute of Infectious Diseases,

Musashimurayama, Japan; 4 Institute for Genetic Medicine,

Hokkaido University, Sapporo, Japan.

A highly attenuated LC16m8 (m8) smallpox vaccine has been

licensed in Japan because of its extremely low neurovirulence pro le,

which was administrated to >100,000 infants where it induced levels

of immunity similar to that of the originating Lister (LO) strain,

without any serious side effects from 1973 to 1975 The m8 was

indirectly isolated from LO through intermediate strains, such as

LC16mO (mO) The m8, a variant that forms small-sized pocks, is a

direct descendant of mO, which itself is a clone that forms

medium-sized pocks, isolated from LO based on its temperature sensitivity

The m8 has lost the function of B5R as the result of a single base

deletion in the ORF, which encodes a 42-kDa glycoprotein that

is essential for formation of extracellular enveloped virus (EEV)

Since EEV is critical for cell-to-cell and long-range spread of the

virus, the B5R dysfunction results in a dramatic reduction of EEV,

and as a consequence, the virus produces small plaques in vitro and

is highly attenuated in vivo We evaluated the oncolytic potential of

m8∆, which is more genetically stable virus by deleting B5R from

m8 that spontaneously generates B5R+ revertants (Kidokoro M

et al., PNAS, 2005), for a variety of different tumor cell lines To

investigate relationship between B5R and oncolytic activity, we also

constructed m8B5R virus from m8∆ by introducing the complete B5R

cloned from mO The m8∆ was able to more ef ciently lyse A549,

BxPC-3 and Caco-2 cells than Huh-7, MDA-MB-231 and SKNAS

cells in culture However, the cytolytic activity of m8∆ was much

lower than that of mO in all kinds of tumors The reduced oncolytic

activity was fully restored by the expression of B5R, as shown in the

cells infected with m8B5R Nevertheless, the m8∆ demonstrated in

vivo oncolytic activity in intraperitoneal xenografts of BxPC-3 cells

stably expressing luciferase Seven days after tumor implantation,

mice received single i.p injection of mO or m8∆ (105, 106, or 107

pfu per mouse) In vivo tumor growth was monitored noninvasively

by bioluminescence imaging after luciferin addition to the treated

SCID mice The bioluminescence imaging on day 11 after treatment

revealed that there was signi cant inhibition of tumor growth in both

mO and m8∆-treated groups, compared with mock therapy group

Quanti cation of this signal indicated that there was no signi cant

difference of tumor volume reduction among mice given 105 pfu and

mice given mO or m8∆ at 10- to 100-fold higher input multiplicities

On the other hand, all mice treated with mO were dead or sacri ced

on days 14 to 21 after treatment because of weight loss and pock

lesions on their tails, paws and in oral cavities In contrast, m8∆

did not induce these viral toxicities, however tumor regrowth was

observed from day 22 post-treatment Our study demonstrated that

m8∆ has the potential of more selective and safer oncolytic agent

than parental virus mO, although it may be necessary to enhance the

antitumor activity

841 Enhanced Tumor Killing by a Mortalin Targeting Modi ed Oncolytic Adenovirus

Jung-Sun Lee,1 Ji Young Yoo,1 Jihoon Ryu,1 Tomoko Yaguchi,2

Sunil C Kaul,2 Renu Wadhwa,2 Chae-Ok Yun.1

1 Brain Korea21 Project for Medical Sciences, Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Shinchon-Dong, Seodaemun-Gu, Korea; 2 Advanced Industrial Science &

Technology, Advanced Industrial Science & Technology, Higashi, Tsukuba, Ibaraki, Japan.

Oncolytic adenoviruses (Ads) offer an effective cancer therapeutic tool with several advantages including wide host cell permeability, high transduction ef ciency, safety, tumor selectivity, non-invasiveness, high genetic modifiability and high level of expression of the integrated transgenes Armed oncolytic Ad in which the therapeutic ef cacy of virus is enhanced by their coupling with cytotoxic, anti-angiogenic or anti-vascular gene products have gained importance

as these engage additional mechanisms for tumor cell killing In this study, we selected mortalin, a stress chaperone that is tightly involved

in human carcinogenesis, constructed a mortalin-targeting oncolytic

Ad (mot-Ad) and examined its therapeutic potential in vitro and in

vivo We demonstrate that the mot-Ad has selective cytotoxicity for

human cancer cells in vitro Retrovirus- mediated overexpression of

mortalin protected the cells against mot-Ad, con rming that mortalin silencing was the real cause of cancer cell death While mortalin overexpression enhanced malignant properties of cancer cells in breast xenograft models, mot-Ad elicited enhanced anti-tumor effect

Immuno-histochemical examination of the tumors showed that the mot-Ad caused enhanced apoptosis and suppression of microvessel formation Since mortalin is upregulated in a large variety of tumors, mot-Ad is proposed as a candidate cancer therapeutic agent

842 Increased Oncolytic Ability of a Double RGD-Modi ed Adenovirus in Ovarian Cancer

Lena J Gamble,1 Qiana L Matthews,1,2 Anton V Borovjagin,3

David T Curiel.1

1 Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL; 2 Center for Aids Research, The University of Alabama at Birmingham, Birmingham, AL; 3 School

of Dentistry, Institute of Oral Health Research, The University of Alabama at Birmingham, Birmingham, AL.

Ovarian cancer is a leading cause of gynecological cancer mortality

in Western countries Attempts to address the urgent need for effective anti-tumor treatments for ovarian cancer patients include development

of oncolytic virotherapy agents targeted speci cally to ovarian cancer cells One major hindrance to effective virotherapy has been suboptimal transduction of ovarian cancer cells due to the sparse presence of the adenovirus receptor on cancer cells Addition of an Arg-Gly-Asp (RGD) motif to the viral binding protein,  ber, increases viral ability to bind and transduce ovarian cancer cells This RGD modi cation has been added to the HI-loop domain of  ber in a virus designed to conditionally replicate in cancer cells, Ad5.∆24RGD The safety of Ad5.∆24RGD has been tested in a Phase I clinical trial Our goal was to improve this anti-cancer agent by further increasing viral infectivity for ovarian cancer cells To this end we created a double-modi ed virus that incorporates an additional RGD double-modi cation

at a separate capsid locale, protein IX (pIX) We have shown that the double-modi ed virus, Ad5.pIXRGD.fRGD, increases ovarian cancer transduction over both of the singly modi ed Ad5.pIXRGD and Ad5.fRGD viruses Based on these data, we hypothesized that a conditionally replicative version of this virus would more ef ciently kill tumor cells than either of the single-modi ed virus controls

Using standard molecular biology techniques we added an RGD-4C sequence to the pIX portion of Ad5.∆24.fRGD to yield, Ad5.∆24

pIXRGD.fRGD Preliminary data indicate that the newly created double modi ed virus incorporates the RGD motif on both the  ber

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CANCER-TARGETED GENE & CELL THERAPY III

and pIX proteins Preliminary data indicates that the oncolytic ability

of Ad5.∆24.pIXRGD.fRGD is greater than that of Ad5.∆24.pIXRGD and trends show that it is at least equal to Ad5.∆24.fRGD in some cell lines and better than Ad5∆24.fRGD in other cell lines We have therefore shown that use of this virus shows proof-of-principle that the pIX protein may be used as a platform for targeting in conjunction with  ber protein on Ad5 Furthermore, use of this virus will not only allow for increased oncolytic capability of the virus as an anti-cancer treatment modality but may also translate to a less toxic potential therapeutic option for the treatment of ovarian cancer

Cancer-Targeted Gene & Cell Therapy III

843 Increasing the Ef cacy of Monoclonal Antibody Therapy of Breast Cancer through Transient Remodeling of Intercellular Junctions

Jonas Persson,1 Max Drescher,1 ZongYi Li,1 Roma Yumul,1 Ying Liu,1 Hongjie Wang,1 Akseli Hemminki,2 Pascal Fender,2 Andre Lieber.1

1 Division of Medical Genetics, University of Washington, Seattle, WA; 2 University of Helsinki, Helsinki, Finland; 3 Institut de Biologie Structurale, Grenoble, France.

The widely used Her2/neu-targeting monoclonal antibody (mAb) trastuzumab (Herceptin(R)) is effective as a single agent in less than 15% of Her2/neu-positive breast cancer patients Like breast cancer, most solid tumors are of epithelial origin A key feature of epithelial cells is intercellular tight and adherens junctions (TJ/AJ)

TJ/AJ represent physical obstacles for tumor access and intratumoral dissemination of anti-cancer therapeutics In studies on breast cancer biopsies, we found that Her2/neu colocalizes to a large degree with TJ/

AJ proteins Based on this, we hypothesize that transient opening of intercellular junctions increases the ef cacy of breast cancer therapy

by trastuzumab We have recently reported that speci c human adenovirus serotypes, namely adenovirus (Ad) serotype 3, 7, 11, and

14 are able to ef ciently infect epithelial cancer cells This is, in part, accomplished by the induction of processes that are reminiscent of Epithelial-to-Mesenchymal Transition (EMT) We demonstrated that the induction of EMT by these Ad serotypes is mediated by viral  ber and penton Speci cally, we showed that recombinant Ad3 penton dodecahedra, i.e particles that spontaneously form when  ber and penton base are expressed in insect cells, trigger the remodeling

of the intracellular junction and the expression/relocalization of mesenchymal proteins within 2 hours upon binding to epithelial breast cancer BT474 cells An effect similar was also observed when BT474 cells were incubated with recombinant Zona Occludens toxin

protein (Zot) derived from Vibrio cholera Importantly, in addition to

remodeling of intercellular junctions, Ad3 PtDd and Zot also induced relocalization of Her2/neu in epithelial cancer cells, resulting in its partial release from trapping in TJ/AJ We  rst tested the effect

of PtDd and Zot on trastuzumab therapy in in vitro models We

demonstrated that trastuzumab in the presence of human complement kills approximately 25% of the Her2/neu positive BT474 cell line

No cell death was observed in Her2/neu negative breast cancer cell lines When BT474 cells were pre-incubated for 2 hours with PtDd or Zot, trastuzumab/complement-mediated cell killing was signi cantly

increased For an in vivo breast cancer model we used BT474-M1

cells injected into the mammary fat pad of CB17/SCID/beige mice

BT474-M1 tumors displayed histological features very similar to

that seen in patients in situ Intraperitoneal trastuzumab injections

into tumor-bearing mice delayed tumor growth Preliminary studies indicate that a combination of intravenous and intratumoral PtDd injection signi cantly enhances the therapeutic effect of trastuzumab

in mice bearing BT474 tumors Zot injection was associated with toxic side effects The PtDd-based approach for transient opening of

intercellular junctions in epithelial cancers has implications for Her2/ neu targeted T-cell therapies and, potentially, for immunotherapies that involve other tumor-associated antigens

844 Improvement of Cancer Gene Therapy by Incorporating a Chicken Hypersensitive Site-4 Insulator into TSTA-Based Tumor-Targeting Vector

Chien-Fu Chen,1 Yu-Ping Sher,2 Mien-Chie Hung,1,3 Chia-Ling Hsieh.1

1 Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University & Hospital, Taichung, Taiwan;

2 Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan; 3 Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX.

Recently, a two-step transcriptional ampli cation (TSTA)-based gene delivery vector in which a tissue-speci c promoter-mediated GAL4-VP16 fusion protein expression cassette was head-to-head connected to a minimal adenoviral E4 promoter containing  ve GAL4 binding sites (G5E4T) for the expression of interesting genes has been developed to augment the transcriptional activity of cellular promoters without impairing tissue-targeting ef cacy Herein, we further improved the gene transduction ef ciency of TSTA vector

by incorporating chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, to alleviate transcriptional interference between cellular and yeast/viral hybrid promoters To select the optimal cHS4 fragment that provides the maximal insulator activity to TSTA vector, we constructed 1.2kb full length, 250bp core, and two tandem repeats of core elements into survivin-VISA-luc plasmid at the site between survivin and G5E4T promoters The effect of cHS4 fragments on transcriptional activity of TSTA system was determined and compared in a survivin-expressing CL1-5 human lung cancer cell line and a survivin-null normal epithelial 184A1 cell line by luciferase assay after plasmid transfection We found that while one copy of core element either within the proximal 250 bp or full length

of cHS4 displayed no signi cant promoting effect on luciferase activity of survivin-VISA-luc vector, around two-fold higher reporter expression was observed by the two tandem repeats of core element

in the orientation forward to G5E4T-luciferase expression cassette

In addition, reverse orientation of cHS4, regardless of the region of insulator, conferred signi cantly higher luciferase expression with a highest induction rate of 4-fold to non-insulated survivin-VISA-luc

by 1.2 kb fragment in CL1-5 cells but remained no effect on 184A1 cells These results demonstrated that cHS-4 insulator in the context

of a TSTA vector can shield a downstream yeast/viral hybrid promoter from upstream cellular enhancers To test therapeutic potential of insulated TSTA vector-mediated cancer gene therapy, we construct

a 1.2 kb cHS4 insulated survivin-VISA-TRAIL vector caring the TNF-related apoptosis-inducing ligand (TRAIL) expression cassette for the treatment of human lung cancer We found that the insulated survivin-VISA-TRAIL vector exhibited more obvious cell-kill effect than non-insulated survivin-VISA-TRAIL in survivin-producing CL1-5 cell line and remain no cytotoxicity in normal 184A1 cells The enhanced cell death was mainly mediated through the robust expression of TRAIL protein that signi cantly triggered downstream apoptotic mechanism, as evidenced by the activation of caspase-3 and binding of annexin V Therefore, we conclude that insulator elements is a useful tool to further improve the ef cacy of currently used TSTA-based tumor-targeting vectors for their promising clinical applications