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CANCER TARGETED GENE THERAPY III

β-galactosidase (Adflt-1LacZ) reporter genes under the control of the

flt-1 promoter to evaluate the transductional activity of this promoter

in SCCHN cell lines in vitro Ad vectors with these reporter genes

under the control of cytomegalovirus (CMV) promoter were used

as control vectors (AdCMVLuc, AdCMVLacZ) RT-PCR and flow

cytometry analyses showed significant transcriptional and

translational activity of the 1 gene in SCCHN cells, whereas

flt-1 promoter activity was relatively high in SCCHN cells, very low

activity was observed in the flt-1 negative control cell lines, HeLa

and normal human keratinocyte, NHEK cells In the analysis with

AdfltLuc or AdCMVLuc, the average relative percentage of flt-1

promoter activity compared to that of the CMV promoter in SCCHN

cells was 32.2%, ranging from 6.76% in FaDu cells to 55.9% in

SCC29 cells Whereas the relative 1 promoter activity in the

flt-1 negative cell lines was 0.098% and 0.flt-136% in HeLa and NHEK,

line infected with AdfltLacZ or AdCMVLacZ; however, HeLa cells

indicate that flt-1 promoter activity is relatively efficient and specific

in SCCHN cells, making it an attractive candidate TSP for SCCHN

cells Driving therapeutic genes or conditionally replicative Ads

with the flt-1 promoter may be useful for the gene therapy of SCCHN

Adenovirus p53 Following Intraperitoneal

Administration in Mice

Kyung Hee Sohn,1 Soon Sun Kim,1 Seung Jun Kwack,1 Rhee Da

Lee,1 Hyun Joo Lee,1 Yong Huck Won,1 Seung Hoon Lee,2 Kui

Lea Park,1 Gyu Seek Rhee.1

1 Reproductive and Developmental Toxicology, National Institute of

Toxicological Research, Seoul, Republic of Korea; 2 Molecular

Therapy center, Sungkyungkwan University, Seoul, Republic of

Korea.

Reproductive and developmental toxicology in the setting of gene

therapy is a relatively new area of investigation but is a central

safety issue for the field An important safety concern of gene

therapy products is the distribution of vector beyond the target

organ This is particularly important if vector distributes to gonads,

raising the possibility of inadvertent germ-line transmission In

addition, for indications such as prostate cancer and ovarian cancer,

the proximity of the point of viral administration to organs of the

reproductive system raises concerns regarding inadvertent

germ-line transmission of genes carried by the virus To evaluate the

reproductive and developmental toxicity of in vivo adenovirus

mediated gene transfer, we studied the biodistribution and potential

germ-line transmission of p53-expressing adenovirus

(Ad-CMV-p53) Both male and female Balb/c mice were injected with 1x108

from major organs including gonadal tissues were analyzed for vector

sequences and expression The PCR analysis showed that there

were detectable vector sequences in liver, kidney, spleen, seminal

vesicle, epididymis, prostate, ovary, and uterus The RT-PCR

were present in spleen, prostate and ovary Vector-administered

female and male mice were mated and their offspring were evaluated

for germ-line transmission of the adenoviral vector The PCR analysis

showed no evidence of germ-line transmission, although vector

sequences were detected in DNA extracted from gonadal tissues

We have also established a ovarian tumor model of human ovarian

cancer in the nude mouse Together, we conclude that the risk of the

inadvertent germline transmission of vector sequences following

intraperitoneal injection of adenovirus is extremely low, although

vector distributed to gonadal tissues

Replicative Adenovirus CNHK300 Targeted to the Telomerase-Positive Cancer Cells

Changqing Su,1 Jonsthon Sham,2 Huibin Xue,1 Mengchao Wu,1

Qijun Qian.1,2

1 Labortory of Viral and Gene Therapy, Eastern Hepatobiliary Surgical Hospital, Shanghai, China; 2 Department of Clinical Oncology, Hong Kong University, Hong Kong, Cape Verde.

Telomerase, a ribonucleoprotein complex mainly composed of the RNA component (hTR) and the telomerase reverse transcriptase (hTERT), plays a crucial role in cellular immortalization and oncogenesis More detailed studies suggested that almost various human primary cancers (>85%) exhibit telomerase activity, but normal tissues do not Thus telomerase may be a promising target for cancer gene therapy Based on our knowledge that hTERT component is a key determinant of human telomerase activity, we constructed a novel replicative adenovirus CNHK300, in which hTERT promoter was introduced three extra E-boxes downstream

of the promoter core sequence and used to regulate E1a gene involved

in adenoviral replication Luciferase assay showed that introducing extra E-boxes into hTERT promoter is beneficial to decreasing the promoter activity in normal cells but does not influence its strong

activity in cancer cells Experiments in vitro and in vivo demonstrated

that, CNHK300 can selectively target to hTERT-positive cancer cells and replicate in them, then result in oncolytic or antitumoral effect, but do not affect the normal cells CNHK300 is superior to ONYX-015 in aspects of selective replication and oncolytic or antitumoral effect These studies suggested that the hTERT promoter-controlled, cancer-selective, replication-competent adenovirus CNHK300 is an ideal vector system for targeted cancer gene therapy

Enhance the Adenovirus Infection of Uro-Epithelium

Nagarajan Ramesh,1 Eric Memarzadeh,1 Melinda VanRoey,1

Virginia Rojas,1 David Frey,1 De-Chao Yu.1

1 Cell Genesys, Inc, Foster City, CA.

Adenovirus has been used widely as an in vitro and in vivo gene transfer vector for the purpose of gene therapy CG8840 is a conditionally replication competent oncolytic adenovirus for the treatment of superficial bladder cancer Intravesicular therapy of refractory superficial bladder cancer employing an adenovirus would allow for local administration and efficient delivery of virus This would reduce the systemic exposure and could allow for repeat treatment of the bladder tumor The glycosaminoglycan (GAG) layer

on the surface of the bladder urothelium has been shown to act as a nonspecific antiadherence factor and may be an important roadblock

to efficient infection of the bladder urothelium by adenoviruses Enhanced infection of bladder urothelium has been achieved in animal models by pretreatment of the bladder or by using specialized adenovirus formulations to disrupt the GAG In order to allow for efficient infection of the urothelium with the oncolytic viruses, we have identified a class of compounds that could be used as a pretreatment agent Preliminary experiments have shown that, at low concentrations, pretreatment with these compounds result in enhanced infection of mice bladder urothelium (>90%) by a replication-defective adenovirus expressing the beta galactosidase gene Of the compounds that were identified, pretreatment with

significant enhancement in the infection of bladder epithelium by adenovirus In a murine model, pretreatment of bladder for 5 min with DDM at a concentration of 0.05% lead to >90% transduction

of the bladder urothelial layer Similar levels of transduction were

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Copyright ®The American Society of Gene Therapy

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CANCER TARGETED GENE THERAPY III

observed even when only 109 viral particles were instilled into the

bladder Only the uppermost epithelial layer was infected by

adenovirus and there was no infiltration of the virus into the inner

layers The integrity of the epithelial layer was not affected following

pretreatment and virus infection Enhancement of adenoviral

infection following pretreatment of bladder with DDM and

oxycholorosene was also observed in a rat model Further work is in

progress to understand the mechanism of action of these chemicals

along with their compatibility for formulation with the adenovirus

Results from these studies will also be presented

Sensitizes Cisplatin-Based Chemotherapy for

Human Head and Neck Cancer

Guoli Shi,1 Guoyan Li,1 Cara Lang,1 Shuzhen Yu,1 Bert

O’Malley,1,2 Daqing Li.1,2

1 Otolaryngology-Head & Neck Surgery, University of Maryland

School of Medicine, Baltimore, MD, United States; 2 Greenebaum

Cancer Center, University of Maryland School of Medicine,

Baltimore, MD, United States.

Chemotherapy plays an important role in the neoadjuvant or

adjuvant setting and management of advanced and metastatic disease

for the treatment of head and neck squamous cell carcinoma

(HNSCC) Cisplatin, a DNA-damaging agent, is one of the most

promising single chemotherapuetic agents used against HNSCC to

date However, the clinical success of Cisplain is compromised if

tumor cells become resistant by various mechanisms Enhanced DNA

double-strand break (DSB) repair could be one of the primary causes

for development of resistance in tumor cells to cisplatin which induce

DNA DSBs or crosslinks Recently, a protein complex made up of

hMre11, hRad50 and Nbs1 (MRN) has been shown to play a critical

component of the cellular response to the repair of DNA DSBs

The present study investigates if the expression of mutant Rad50

by a dominant negative adenoviral construct in tumor cells inhibits

the function of MRN and increases sensitivity to Cisplatin-based

chemotherapy against human HNSCC

Human HNSCC cell lines were used in all experiments A dominant

negative recombinant adenovirus containing a gene coding for the

mutant Rad50 protein under transcriptional control of a

cytomegalovirus promoter (Ad-Rad50) was constructed Control

adenovirus DL312 with E1a region deletion was obtained from Dr

Tom Shenk, Princeton University Cisplatin was purchased form

Bristol-Myers Squibb Co Expression of adenovirus-mediated Rad50

was confirmed by Q-RT-PCR Cell growth curves were determined

by MTT Q-RT-PCR analysis confirmed the expression of mutant

Rad50 expression after Ad-Rad50 gene transfer in HNSCC cells In

multiple trials, cell growth curves were seen to flatten and decline in

combined Ad-Rad50 and Cisplatin treated group as compared to

the control groups in which cellular division continued at a brisk

pace Our current study indicates that alteration of DNA repair

may provide a novel approach to enhance sensitization of HNSCC

to chemotherapy and support the potential application of Ad-Rad50

in combination of cisplatin to treat advanced and metastatic HNSCC

(CAR) Expression in the Non-Neoplastic Genito-Urinary Tract: Implications for Gene Therapy Using Adenoviral Vectors

Jeff Simko,1 Vivian Weinberg,1 Peter R Carroll,1 Frank McCormick,1 Katherine A Rauen.1

1 Comprehensive Cancer Center, University of California San Fransisco, San Fransisco, CA, United States.

Adenoviral-based gene therapies are dependent on infectivity of the target tissue through the coxsackie-adenovirus receptor, CAR, which is the primary adenoviral receptor Its presence is a determining factor in efficient adenoviral infection CAR serves as the site for attachment of human adenovirus via the fiber protein to the target cell membrane CAR is found in the tight junctions between epithelia cells and its physiologic function is believed to be important in cell-cell interaction and adhesion We have recently demonstrated that metastatic prostate carcinoma expresses high levels of CAR, especially relative to primary tumors confined to the prostate (Rauen

et al., Cancer Research 62: 3812 2002) While studies of CAR membrane expression levels in other tumors of the genito-urinary (GU) tract is ongoing, we have evaluated the benign epithelia of the male GU system to obtain a baseline of CAR expression in these tissues The rationale for the study is several fold: to examine the normal physiologic expression of this cell adhesion molecule so as

to provide a baseline for comparing relative CAR expression levels between normal and tumor tissue, to understand how much virus may be absorbed by the benign tissue component during therapy and to know whether toxicity of normal tissues may be a serious consideration due to possible infectivity We evaluated CAR expression in normal formalin-fixed, paraffin-embedded archival tissues of the male GU system which included prostate (n=33), kidney (n=15), ureter (n=9), urinary bladder (n=12), seminal vesicle (n=6), urethra (n=8) and testis (n=10) The intensity of CAR staining for the normal specimens were grouped into four categories: no staining/background of negative controls (0), weak staining detectable above background (1+), moderate staining (2+), and intense staining (3+) All prostate, bladder, kidney, ureter and testis tissues demonstrated strong 2-3+ staining Reduced staining (1 – 2+) was present in all seminal vesicle and urethral specimens These results indicate that the intrinsic expression level of CAR throughout the epithelia of the GU tract is high, suggesting that these tissues are potential targets for infectivity

Adenovirus Targeting of Gliomas

Winan J Van Houdt,1,3 Joel N Glasgow,1,2 Hongju Wu,1,2

Xiaosheng Lei,1,2 Martine L M Lamfers,3,4 Clemens M F Dirven,4 David T Curiel,1,2 Yosef S Haviv.1,2

1 Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery; 2 Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL; 3 Department of Medical Oncology, Division of Gene Therapy; 4 Department of Neurosurgery, VUMC, Amsterdam, Netherlands.

Malignant gliomas are resistant to radiotherapy and chemotherapy Because of the high mortally rates in patients with malignant glioma, new treatment strategies are required Therefore, gene therapy approaches have been undertaken, to the end of introducing novel mechanisms of glioma cell killing Adenoviral vectors (Ads), based

on serotype 5 (Ad5), have been widely used for gene delivery in different cancer types However, a limiting factor for the utility of Ad5 vectors for cancer gene therapy is their critical dependence on the membrane expression of the primary Ad receptor, the coxsackie and adenovirus receptor (CAR) Most tumor cells, including most glioma cells, express CAR at low levels Thus, the efficacy of Ad5