812 design and hydrodynamic gene transfer of ‘sticky’ IFNγ for liver directed gene therapy of hepatic metastasis of cancer cells

812 Design and Hydrodynamic Gene Transfer of ‘Sticky’ IFNγ for Liver Directed Gene Therapy of Hepatic Metastasis of Cancer Cells Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © Th[.]

Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy

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CANCER-TARGETED GENE & CELL THERAPY III

810 Down-Regulation of Breast Cancer

Resistance Protein (BCRP) by siRNA Delivery

Using Lipid-Substituted Aliphatic Polymers

Hamidreza Montazeri Aliabadi,1 Breanne Landry,1 Parvin

Mahdipoor,1 Hasan Uludag.1

1 Department of Chemical & Material Engineering, University of

Alberta, Edmonton, AB, Canada.

Polycationic polymers have been promising carriers in siRNA

delivering strategies However, their potential has been limited by

unacceptable toxicities and/or inadequate effi cacy Hydrophobic

modifi cation with lipophilic moieties is an effective approach to

enhance the siRNA delivery effi ciencies, and this study methodically

evaluated the effect of a range of lipophilic substitutions on low

molecular PEI for siRNA delivery against a major effl ux protein

involved in multidrug resistance, Breast Cancer Resistance Protein

(BCRP) Although BCRP was discovered in a breast cancer cell line,

this membrane protein is expressed in different leukemia and solid

tumor cells The BCRP has been additionally proposed to play a

key role in chemoresistance in cancer stem cells Therefore, specifi c

down-regulation of BCRP is a promising approach in sensitizing

cancer cells to chemotherapeutic agents In this study, we synthesized

a series of hydrophobically modifi ed 2 kDa PEIs by using different

lipophilic moieties and evaluated their characteristics and potential

as a siRNA delivery system for protein down-regulation Lipid

substitution increased the toxicity of PEI2 for some of the higher

lipid substitutions; however, this toxic effect was signifi cantly less

than the toxicity displayed by the 25 kDa branched PEI routinely

used for nucleic acid delivery A signifi cant increase in siRNA cellular

uptake in wild-type and transfected BCRP-positive MDCK cells

was observed with polymer:siRNA ratios of 1:2 and 1:8 when

lipid-substituted polymers were used The extent of lipid substitution was

directly proportional to the extent of siRNA delivery into the cells

A signifi cant down-regulation in the target protein expression was

also detected after 48 hours using 9-36 nM siRNA (complexed with

lipid-substituted polymers), with a linear dose-effect relationship

in the evaluated concentration range The highest down-regulation

effect was achieved with Linoleic Acid-substituted polymers, and a

strong correlation was observed between the siRNA uptake and

down-regulation level BCRP down-down-regulation after treatment with 36 nM

siRNA was shown to sensitize the drug-resistant cells to cytotoxic

effect of mitoxantrone by a 14-fold drop in the IC50 of the drug in the

siRNA treated resistant cells

This effect started to “wear off” after 7 days Overall, this study

demonstrated the possibility of a safe and effective siRNA delivery

to cancer cells by hydrophobically-modifi ed low molecular PEI We

also demonstrate here the importance of the polymer structure in

determining its performance as a nucleic acid delivery system, as

well as introducing a line of safe and effective polymers that could

be designed for optimal effi cacy

811 Experimental Gene Therapy of Nasopharyngeal Carcinoma Targeted to Telomerase

Wen Zhong.1

1 Dept of ENT, Dept of ENT, ZhuJiang Hospital, Guangzhou City, China.

Telomerase is closely related to tumorigenesis As an ideal target, regulating telomerase activity has become a new direction for cancer gene therapy TK suicide gene vectors with hTERT single promoter and hTERT/CMV double promoter as well as expression vector of telomerase inhibitor PinX1 are able to target to telomerase and kill

nasopharyngeal carcinoma (NPC) in vitro and in vivo In this study, we

constructed three expression vectors: hTERT-TK, CMV/hTERTp-TK and pEGFP-C3-PinX1, and studied their tumor inhibitory effects on NPC 5-8F cells and in NPC xenograft in nude mice Expression of

TK was confi rmed by immunostaining, fl uorescent quantitative PCR, telomerase activity assay and fl ow cytometry The results indicates that TK expression in cells transfected with CMV/hTERTp-TK was

2∼5-fold of that in cells transfected with hTERTp-TK Consequently, transfection of CMV/hTERTp-TK significantly inhibited NPC viability and migration as examined by MTT assay and Boyden chamber invasion experiment, respectively, and attenuated growth of NPC exnograft in nude mice compared to transfection of

hTERTp-TK (p<0.05) Pathological examination of liver and kidney of the nude mice showed no obvious side effects Taking together, the data suggested that the CMV/hTERTp-TK has improved anti-tumor effect and similar gene targeting ability and safety to hTERTp-TK

In addition, transfection of pEGFP-C3-PinX1 in NPC signifi cantly enhanced PinX1 mRNA level, while decreased hTERT mRNA level

by 21% (P <0.05) and telomerase activity (p<0.05) compared with nontransfected cells Moreover, overexpression of PinX1 in NPC inhibited tumor growth, reduced cell migration and wound-healing ability, arrested cells in G0/G1 phase and induced cell apoptosis (apoptotic index was 49.73±2.70%, p<0.05) Furthermore, those effects were reversed by transfection of PinX1-FAM-siRNA, which decreased PinX1 mRNA level by 70% In summary, the above experiments clearly indicate that cancer gene therapy specifi cally targeted to telomerase can effectively inhibit telomerase activity in

NPC and tumor cell proliferation both in vitro and in vivo.

812 Design and Hydrodynamic Gene Transfer

of ‘Sticky’ IFN γ for Liver-Directed Gene Therapy of

Hepatic Metastasis of Cancer Cells

Mitsuru Ando,1 Yuki Takahashi,1 Hanae Mukumoto,1 Makiya Nishikawa,1 Yoshihiko Watanabe,2 Yoshinobu Takakura.1

1 Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Science, Kyoto University, Kyoto, Japan; 2 Department of Molecular Microbiology, Graduate School of Pharmaceutical Science, Kyoto University, Kyoto, Japan.

Purpose: Interferonγ (IFNγ) has been expected to be applied for the treatment of several diseases, including cancer and hepatitis C virus infection We have demonstrated that gene transfer of murine IFNγ is effective in inhibiting tumor metastasis and the onset of atopic dermatitis To increase its therapeutic index, controlling the tissue distribution of the cytokine after in vivo gene transfer should

be important because its multifunctional nature causes several side-effects The purpose of this study is to limit the distribution of IFNγ expressed in the liver to this organ To achieve this, we designed a

‘sticky’ derivative of IFNγ and delivered plasmid DNA expressing

it to mouse liver The effects on hepatic metastasis and systemic

side-effects were evaluated in mouse models Methods: One to

three repeats of the heparin binding domain (HBD) of extracellular superoxide dismutase were genetically fused to the C-terminal of

Molecular Therapy Volume 19, Supplement 1, May 2011

Copyright © The American Society of Gene & Cell Therapy S311

CANCER-TARGETED GENE & CELL THERAPY III

murine IFNγ Plasmid DNA expressing the fusion protein,

pCpG-IFNγ-(HBD)n (n=1 to 3), was delivered to mouse liver through

hydrodynamic injection The serum concentration of IFNγ was

determined by ELISA, and its biological activity in the liver was

determined by measuring the mRNA levels of IFNγ-inducible

molecules M5076 murine ovarian sarcoma cells were inoculated to

mice via the tail vein to establish a mouse model of hepatic metastasis,

and the survival of mice was monitored The toxicity was evaluated

by anorexia, body weight loss and liver damage Results: The mRNA

expression of IFNγ derivatives decreased with increasing number of

HBD on IFNγ The serum concentration of IFNγ also decreased with

the HBD number, and a large decrease was observed for IFNγ-(HBD)2

and IFNγ-(HBD)3, suggesting that two or more repeats of the HBD

are effective in capturing the fusion proteins onto liver cells An

intravenous injection of heparin resulted in a transient increase in the

serum concentration of IFNγ-(HBD)2 Despite the huge differences

in the serum concentration of IFNγ, the ratio of the mRNA levels of

IFNγ-inducible molecules to those of IFNγ in the liver was almost

comparable to all groups A hydrodynamic injection of pCpG-IFN

γ-(HBD)2 was as effective as that of pCpG-IFNγ in suppressing the

liver metastasis, whereas it caused no signifi cant liver toxicity, loss

of body weight and anorexia, which were obvious in mice receiving

pCpG-IFNγ Conclusion: We have demonstrated that gene transfer of

IFN-γ-(HBD)2 to the liver is effective in inhibiting hepatic metastasis

as well as in reducing IFN-γ-inducible systemic side-effects

813 Cytokine-Based Log-Scale Expansion of

Functional Human Dendritic Cells from PBMCs

Yui Harada,1,2 Satoru Saito,1 Yosuke Morodomi,1 Kumi Yoshida,1

Tomohiko Ichikawa,2 Yoshikazu Yonemitsu.1

1 R &D Laboratory for Innovative Biotherapeutics, Kyushu

University Graduate School of Pharmaceutical Sciences, Fukuoka,

Japan; 2 Departments of Urology, Chiba University Graduate

School of Medicine, Chiba, Japan.

PURPOSE: Dendritic cells (DCs) play a crucial role in maintaining

the immune system Though DC-based cancer immunotherapy

has been suggested to hold potential to treat various kinds of

malignancies, clinical effi cacies are still insuffi cient in many human

trials We proved that this antitumor effect depends on the number

of DCs (unpublished data), and it is necessary to prepare an enough

number of DCs for effective treatments of tumors In this study,

therefore, we attempted to expand functional human DCs ex vivo

with cytokine cocktail Materials and Methods: Peripheral blood

mononuclear cells (PBMCs) and CD14+ cells were obtained from

volunteers Conventional DCs were obtained from peripheral blood

CD14+ cells as described previously with minor modifi cation Briefl y,

CD14+ cells were cultured under hGM-CSF and hIL-4 (GM/IL-4)

CD3-depleted PBMCs were expanded and differentiated into DCs

in the presence of hGM-CSF and hSCF (GM/SCF) or GM/IL-4 for

several weeks Expanded DC properties such as expression of surface

markers, infl ammatory cytokines production, phagocytotic activity,

antigen presentating ability in vitro were analyzed, and compared

with those of conventional DC RESULTS: CD3-negative cells

increased approximately 10 - 100 fold after 5 weeks culture and

>80% of expanded cells expressed CD11c Thus, by this method, 10

- 100 times more CD11c+ cells could be obtained than conventional

procedures could As are seen in conventional DCs, expanded

DCs showed dendrites after maturation, and endocytotic activities

Expanded DCs also expressed HLA-DR, adhesion molecules, and

co-stimulatory molecules and produced infl ammatory cytokines

as well as conventional DCs did Functionally, mixed lymphocyte

reaction (MLR) assay revealed that expanded DCs could stimulate

allogenic T-cell proliferation to the same extent as conventional DCs

CONCLUSIONS: We found that human CD11c+ cells could be

effectively expanded from PBMCs by culture with cytokine cocktail

(GM/SCF) Expanded DC had properties that were required to obtain therapeutic gain We expect that this technology will be able to contribute largely to both basic and clinical research of human cancer immunotherapy DC expansion technology will improve therapeutic gain of cancer and alleviate patients’ burden of apheresis

814 A New Method To Develop Genetically Modifi ed Lung Cancer Stem Cells

Feridoun Karimi-Busheri,1 Victoria Zadorozhny,1 Ali Haghighi,1 Daniel L Shawler,1 Habib Fakhrai.1

1 NovaRx Corporation, San Diego, CA.

Discovery of cancer stem cells has initiated a new era that holds much promise and hope in the fi ght against cancer Tumor stem cells are resistant to chemotherapy and radiation and are reputed to be one

of the major causes of cancer recurrence We have established an intense program to better understand lung cancer tumor stem cells and develop new therapeutic approaches against lung cancer, the most prominent, and most common, cancer killer in the world The annual death due to this disease exceeds the combined deaths caused

by colon, breast, prostate, and pancreatic cancers Thus, development

of an effective therapy is critical to serve this serious unmet medical need Lung cancer causes sever morbidity and mortality and its cost

to the society related to treatment costs, morbidity, and mortality approaches a 100 billion annually in the United States Here we report the isolation and gene modifi cation of lung cancer stem cells from established non-small cell lung cancer (NSCLC) cell lines and from primary cells of a NSCLC tumor xenograft established in NOD/SCID mice We also present methodologies towards genetically engineering the isolated cells by antisense gene modifi cation with the TGF-β antisense vector pKNC/SBA2 and the generation of large-scale quantities of the gene modifi ed tumor stem cells for utilization

in clinical trials Our data demonstrate that the secretion of TGF-β

is signifi cantly reduced by the transformed vector and expression of many genes is signifi cantly altered in gene modifi ed cells compared to the parental unmodifi ed cell lines Inclusion of a specifi c cancer stem cells vaccine as components of our whole tumor cell vaccine cocktails belagenpumatucel-L, which has shown effectiveness in 70 percent of the patients in a phase II clinical trial, may result in an even higher survival among the patients and decreasing tumor metastasize

815 miRNA from Tumor-Associated Macrophages as Serum Biomarkers for Ovarian Cancer

Jonas Persson,1 Ines Beyer,1 Nicole Urban,2 Charles Drescher,2 Andre Lieber.1

1 Division of Medical Genetics, University of Washington, Seattle, WA; 2 Fred Hutchinson Cancer Research Center, Seattle, WA.

Our fi ndings and published data show that ovarian cancers are heavily infi ltrated by tumor-associated macrophages (TAMs) TAMs secrete factors that support tumor growth and prevent anti-tumor immune responses There is a clear correlation between the number

of TAMs and malignancy Our central hypothesis is that TAM derived miRNA can serve as a serum biomarker for cancer miRNAs have distinct expression profi les in different tissues They are actively released from cells and highly stable in a cell-free form in the blood

To test our hypothesis, we isolated TAMs from mouse tumors or biopsies from ovarian cancer patients and compared their miRNA expression profi les with those of peripheral blood monocytes from animals or patients without tumors Based on this, we selected a set of miRNA that were expressed at a signifi cantly higher level in TAMs In preliminary studies in mice, we found that several of these miRNAs were present at signifi cantly lower levels in serum from mice with tumors compared to serum from control mice Furthermore, the concentration of these miRNA inversely correlated with the tumor