733. Comparative Evaluation of oncolytic Adenoviruses for Selective Eradication of oral Cancer and Precancerous Lesions

of addressing both the CD133+ cancer stem cells and the CD133' cells of the tumor. In particular, Ad16 and CV23 are meeting this challenge.

731. Carrier Cell-Mediated Delivery of a Replication-Selective Oncolytic Adenovirus for Ovarian Cancer Gene Therapy

Katsuyuki Hamada,' Yaka Hattori.!Ting Zhang,"AyakoNagai;' Michihiro Sumida.'

'Obstetrics and Gynecology. School ofMedicie, Ehime University, Toon, Ehime, Japan; lEngineering f or Production and Environment, School ofScience and Engineering. Ehime University, Matsuyama, Ehlme, Japan; 'Oral and Maxillofacial Surgery; School ofMedicine, Ehime University. Toon, Ehime, Japan; "Geriatric Medicine. School ofMedicine. Ehime University. Toon, Ehime, Japan; 'Basic Research and Education ofMedical Science. School ofMedicine. Ehime University, Toon, Ehime, Japan

Although replication-competent viruses have been developed to treat cancers,their cytotoxiceffectsare insufficient, since infection with them is inhibited by generation of neutralizing antibodies. To address this limitation, we developed a carrier cell system to deliver a replication-competent adenovirus. Carrier cells infected with replication-competent adenovirus were incubated with target ovariancancer cells in high titer of antiadenovirusantibody. Carrier cells wereinjectedintosyngeneicsubcutaneous ovariantumorsafter immunization with adenovirus. Carrier cell-derived cell fragments containingviral particleswere engulfedby proliferative targetovarian cancer cells. This engulfment-mediated transfer of adenovirus was not Inhibited by antiadcnovirus antibodyand enabledrepetitive infection, After the induction of antiadcnoviral CTL responses by immunization of adenovirus, administration of carrier cells infected with a replication-competent adenovirus induced complete tumor regression. Adenovirus-GM-CSF augmented the antitumor effect of carrier cells by increasing antiadenoviral and antitumoral CTL responses and decreased the number of injections of carrier cells required to induce complete tumor regression. This novel carrier cell-mediated viral transfcctionsystemmightproveuseful incancer gene therapy.

732. Development of Novel Hybrid AdenoSemliki Forest Viral Vectors for Anti-Cancer Applications

Michael L. Roberts,I Polyxeni Katsoupi,' Teni Boulikas.' 'Gene Therapy Research, Regulon Inc.• Mountain View. CA: lMolecular Biology. Regulon A.E., Athens, Attiki, Greece.

The development of effective gene transfer vectors is essential if gene-based therapeuticswill succeed in the e1inic. Vectors based on viral systems remain the most efficient means of introducing exogenous DNA into target cells, however, no one virus retains all the attributesrequiredto mediateefficientgene expression for therapeutic application.Adenoviral vectors are the most widely applied vectorsystem in pre-clinical and clinicalsettings, given their easeof manipulation, hightiterproduction, highcapacity, broadtropismand generalsafety profile. Semliki ForestViral(SFV) vectors represent a less widely used expression system that is capable of generating very high levels of target protein in infected cells. We have previously constructedtemperaturesensitive SFV vectorsharboringtwo point mutations in the viral nsP2 gene that resulted in an increased safety profile, associated with prolongedgene expression in target cells. We have further developed this tsSFV mutant vector into a two-layered expression system, whereby the SFV replicon is first expressed from a CMV promoter in situ that subsequently allows for the expressionofEGFP reportergene fromthe SFVsubgenomic promoter. Usingthis system we have observedexpressionofEGFP S280

to levelstwoordersof magnitudehigherthanthosewitnessedfrom a simpleCMV-EGFPexpression cassette(see figure). We have further generated E4-deleted second-generation adenoviral vectors based on this two-layered tsSFV system, where the initial expression of the SFV replicon is conditionally expressed in order to facilitate efficientadenoviralvectorproductionand reducepotentialtoxicity. We present data demonstrating increased stringency and efficacy of transgene expression compared to standard adenoviral-based systems and its further application in the expression of novel anticancer genes. FACS Anii

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733. Comparative Evaluation of oncolytic Adenoviruses for Selective Eradication of oral Cancer and Precancerous Lesions

Hester J. T. van Zecburg,' Ruud H. Brakcnhoff,' Aafke Huizenga,' Zeng B. Zhu,"Victor W. van Beusechem.'

'Otolaryngology/Head-Neck Surgery, Section Tumor Biology. VU University Medical Center; Amsterdam, Netherlands: 2Department ofPathology; University ofAlabama at Birmingham, Birmingham ; "Department Medical Oncology. Division Gene Therapy, VU University Medical Center; Amsterdam, Netherlands.

Eachyear over 270,000patientswith oralsquamouscell carcinoma (OseC) are diagnosed worldwide.The tumorsarc mostlytreated by surgery and postoperative radiotherapy, and despite advances in treatment, the long-term survival rates of patients with OSCC have only marginallyimprovedduring the last 20 years. OSCC arc preceded by preneoplastic lesions in the oral mucosa that are often not completely excised causing second field tumors. Mouthwash therapy with oncolytic adenoviruses might be considered an option for treatment of oral precursor lesions. In this study, we tested nine oncolyticadenoviruseswithdifferent EI deletionsor selective promoters driving EIA expression, with an infectivity-enhancing capsid modification, and/or p53 transgcne expressionfor their lytic replication in fiveHNSCCcell linescomparedto two non-malignant cell types (i.e., keratinocytesand fibroblasts). To this end, viruses were addedto the cells in a two-folddilutiontitration in the rangeof 1,000to 0.0012 IU/cell in triplicate. After 7 days, cell viabilitywas analyzed by measuring WST-I conversion. The 50% tissue culture inhibiting dose (TCID50) was determined and normalized by the TCID50 of wild-type Ad5. Average normalized TCID50 values were calculatedfrom at least three independent assays on each cell line. To determine cancer-selective toxicities, selectivity indexes (SI) were calculated as the ratios of normalizedTCID50 values of Molecular Therapy Volume 15.Supplement t, .\by 2007 Copyright © '111C American Society of Gene Therapy

HNSCC cells over normal cells, both keratinocytcs and fibroblasts. Interestingly, this yielded quite differentresults for keratinocytesand fibroblasts. SI (HNSCC/fibroblast) values were highest for AdD24 (SI=2.4) and AdD24-p53 (SI= I I) viruses. Keratinocytes were very sensitive to all test viruses, but Onyx-OI5, Ad5-D24RGD and two viruses with selective (CXCR4 and Survivin) promotersdriving E IA gave SI (HNSCC/keratinocyte) values above I. The virus with the Survivin-driven EIA showed most favorable results (SI=17).Our results indicate that conditionally replicative adenoviruses might be suitable for treatment of oral precursor lesions. Surprisingly, keratinoeytes and fibroblast behaved very dilTerently in terms of sensitivity to oncolytic adenovirus replication. Selectivity indexes should for that reason be determined on both cell types. Ofthe nine oncolytic adenoviruses tested, the virus with Survivin-drivcn EIA appeared most promising in relation to normal oral keratinocytes, whileAdD24-p53appeared most promising in relationto fibroblasts. From the current comparison we cannot deduce unequivocally which virus modification provides the most selective elTect. Since keratinoeytes are a major constituent ofthe oral mucosa, a potential concern is the high sensitivity of normal keratinocytes for most of the tested viruses.

polyphosphoramidatc (PPA). This study represents a sensitive and quantitative method to evaluate trafficking an~ unpacking be~aviors of DNA nanocomplexes intracellularly, settmg the foundation for the development of a relevant kinetic model that may be applied to gene carrier optimization.

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Hunter H. Chen.l -' Vi-Ping Ho,' Xuan Jiang," Hai-Quan Mao,' Tza-Huei Wang,' Kam W. Leong." I Biomedical Engineering, Johns Hopkins University, Baltimore, MD; 2Biomedicaf Engineering, Duke University, Durham, NC; "Meciumtcal Engineering, Johns Hopkins University, Baltimore, MD; "Materials Science and Engineering, Johns Hopkins University. Baltimore, MD. Cationic polymers that condense plasmid DNA through electrostatic interactions to form nanocomplexes have emerged as safer, though less efficient, options than viral vectors for gene transfer. Rational design ofmore efficient gene carriers will be possible only with mechanistic insights of the rate-limiting steps in the nonviral gene transfer process. In order to examine the intracellular transport and unpacking of polymeric DNA nanoeomplexes for gene therapy, their component plasmid DNA and polymers were individually labeled with quantum dots (QDs) and fluorescent organic dyes, respectively, as a donor and acceptor pair for fluorescence reson~n~e energy transfer (FRET) (Fig. I). The high signal-to-noise rauo m QD-mediated FRET (QD-FRET) enables sensitive detection of discrete changes in nanocomplex state at the single-particle level. Because FRET is abrogated when a nanoeomplex is dissociated, it can be considered as a digital (on/oft) indicator of DNA release. The photostability of QDs allowed further intracellular and intranuelear tracking of the free QD-Iabeled plasmids. The size and zeta-potential of QD-FRET nanocomplexes , as determined by dynamic light scattering, were not significantly affected upon labeling; thus, they behave similarly to unlabeled nanocomplexes. The intracellular uptake and unpacking of nanocomplexes through QD-FRET was captured over time by confocal microscopy. At each time point, quantitative three-dimensional analysis ofthe collected image stacks provided the fractional distributions of both stable nanocomplexcs and released plasmid DNA within each of three major cellular compartments: endosomes/lysosomes, cytoplasm, and the nucleus. The intracellular trafficking and DNA release kinetics were compared among nanocomplexes formed from different polymeric gene carriers: chitosan, polyethylenimine (PEl), Molecular Therapy Volume 15.Supplement I. ~br Copyright © The American Soc iety o f Gene Therapy

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734. Quantitative Analysis of Intracellular Unpacking of Polymeric DNA Nanoparticles Constructed from Quantum Dot-FRET

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735. Design of Peptide Vinyl Ester Inhibitors of the Proteasome To Increase Gene Transfer Efficiency

Molly E. Martin, Kevin G. Rice. 'Mediclnal and Natural Products Chemistry; University ofIowa, Iowa City; IA.

The proteasome is a multisubunit complex that is responsible for the degradation of many cytosolic proteins. The 26S protcasomc complex is composed of the barrel-like 20S catalytic core capped at the ends by the 19S regulatory subunits. The 19S regulatory particles are responsible for substrate recognition, unfolding, a~d translocation of proteins into the catalytic core. The proteolytic sites of the proteasome arc contained inside the core of the 20S complex. Unlike many other proteolytic enzymes, the proteasomc has multiple peptidaseactivities that can be elassificdinto three main groups: cleavage after hydrophobic side chains (chymotrypsin-li~e), cleavage after basic residues (trypsin-like), and cleavage after aCIdIC residues (peptidylglutamyl peptide hydrolysis or PGPH). Previ?us studies have shown that the proteasome is a key route of'metabolism for peptide-basednon-viralgene delivery systems.1 The gene transfer efficiency of peptide-DNA condensates can be enhanced by the addition ofcommercially available proteasome inhibitors that prevent premature degradation of the gene delivery peptide. Peptide vinyl esters arc a new elass of proteasome inhibitors that arc suggested to work through a mechanism similar to the well-known peptide vinyl sulfone inhibitors, such as NLVS. The C-terminal vinyl e~ter covalently modifies the active site of the proteasome by serving as a substrate for a Michael addition with the catalytic threonine residue at the Nvterminusof the 13 subunit. Wehypothesize that gene delivery peptides containing a Csterminal vinyl ester moiety may resist metabolism by the proteasome and thereby increase the gene transfer efficiency of peptide-DNA condensates. Here we describe the synthesis and testing of gene delivery peptides containing a variety of C-terminal peptide vinyl ester sequences that condense DNA, mediate cellular uptake, inhibit the proteasome, and boost gene transfer efficiency. Incorporation of the peptide inhibitor seS281