167. Polyethyleneimines as Vehicles for Oral Gene Delivery

OTHER DNA VIRUSES and whole-cell electrophysiology. Ca+2 influx following TRPV1 activation by known agonists resulted in mitochondrial permeability transition and subsequently initiated an apoptotic cascade leading to cell death. Since cellular apoptosis occurred prior to completion of the virus replicative cycle, TRPV1 activation indirectly impaired HSV-1 vector replication, resulting in a fall of viral titers. An inclusion of known TRPV1 antagonists prevented calcium influx through TRPV1, consequently reversing this blockage and allowing vector replication to occur in the presence of TRPV1 agonists. This growth recovery of HSV-1 vectors following TRPV1 inactivation provides the basis for a selective vector replication-based assay to identify TRPV1 antagonists. Studies with mixed virus infections demonstrate the sensitivity of this system and allow the selection of 1 viral particle among 100,000 background virions. The HSV-1 vector-based selection assay described herein should provide a powerful approach for identifying novel gene products that regulate TRPV1 function from a cDNA library cloned into the TRPV1-expressing backbone HSV-1 vector. This approach also offers advantages of (i) efficient and flexible transgene expression in a variety of cell types and (ii) ease of adaptability to high throughput formats.

165. Enhanced Baculovirus-Mediated Transduction of Human Cancer Cells by TumorHoming Peptides Anna R. Makela,1 Heli Matilainen,1 Daniel White,1 Christian Oker-Blom.1 1 NanoScience Center, University of Jyvaskyla, Jyvaskyla, Finland. Tumor cells and tumor vasculature both offer specific molecular targets that can be utilized for site-directed delivery of therapeutic genes. In order to achieve tumor-specific gene delivery, the tropism of baculovirus can be manipulated by modification of the virus envelope using baculovirus display technology. We genetically engineered a series of recombinant display viruses by fusing various tumor-homing peptides to the transmembrane anchor of vesicular stomatitis virus G-protein to establish more efficient and selective baculovirus vectors for targeted gene delivery into human cancer cells. The viruses were further equipped with a luciferase expression cassette, allowing transduction monitoring in mammalian cells by measuring luciferase activity. The fusion proteins were successfully incorporated into budded virions and these modified viruses showed significantly improved binding to both human breast carcinoma (MDA-MB-435) and hepatocarcinoma (HepG2) cells as measured by flow cytometry. Binding of the tumor-homing peptide displaying viruses to target cells was reduced to the level of the control virus by preincubating the cells with the corresponding soluble peptides. Moreover, a maximum of 7- and 24-fold increase in transgene expression was achieved for these cell lines, respectively. The enhanced binding and transduction strongly suggest that the displayed peptides dictated this behavior. Together, these results imply that the specificity and efficiency of baculovirus-mediated gene delivery can be notably enhanced in vitro when tumor-targeting ligands are used and therefore also highlight the potential of baculovirus vectors in cancer gene therapy in vivo.

166. OncoVEX: A Family of Oncolytic Herpes Simplex Viruses Optimised for Therapeutic Use Robert S. Coffin,1 Binlei Liu,1 Han Ziqun,1 Magdalena Assenberg,1 Suzanne Thomas,1 Jennifer Hu,2 Guy Simpson.1 1 Research, BioVex Inc., Cambridge, MA; 2Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom. HSV in which the neurovirulence factor ICP34.5 is inactivated directs tumour selective cell lysis and has proven safe in the Phase I clinical studies conducted so far. To produce oncolytic HSV with S64

enhanced anti-tumour properties, we have deleted ICP34.5 from a clinical isolate of HSV-1, which enhances the tumour cell killing capabilities of the virus, and also deleted ICP47 (which blocks antigen presentation in HSV infected cells) and inserted the gene encoding GM-CSF. This aimed to maximize anti-tumour immune responses following intra-tumoural injection and provide an in situ, patientspecific, anti-tumour vaccine, combined with oncolysis. In vivo, both injected and non-injected tumours could be cured with this virus and animals were then protected against further tumour cell challenge. Following this promising data a Phase I clinical trial with the virus (OncoVEXGM-CSF) was conducted including patients with cutaneous or sub-cutaneous deposits of a number of tumour types (Lead Investigator: Professor Charles Coombes, Hammersmith Hospital, London). This demonstrated the virus to have a good safety profile, the main side effects observed being ‘flu-like symptoms, similar to those which have previously been observed with other oncolytic products and cancer vaccines, combined with evidence of biological activity including virus replication and GMCSF expression. In addition inflammation, tumour flattening and necrosis was observed in injected lesions which was in some cases considerable and which was also in some cases observed in lesions which had not themselves been injected. In all cases where necrosis was observed in biopsy material, this correlated with areas of staining for HSV, suggesting the virus had caused the effect. Following this promising data, preparation for Phase II studies is underway. In addition to OncoVEXGM-CSF, further versions of the OncoVEX virus expressing other active genes have been constructed and tested in pre-clinical models. These include a virus expressing TNF∝, intended to be synergistic with radiotherapy, and versions of the virus expressing a pro-drug activating gene combined with the delivery of a fusogenic glycoprotein designed to maximize the properties of the virus for local tumour control. Each of these have shown promising results in pre-clinical tumour models, including in combination with chemotherapy and radiotherapy where benefits which are at least additive have been demonstrated.

NON-VIRAL GENE DELIVERY 167. Polyethyleneimines as Vehicles for Oral Gene Delivery Eytan A. Klausner,1,2 Kam W. Leong.1,3 Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD; 2Department of Pharmaceutical Sciences, Midwestern University Chicago College of Pharmacy, Downers Grove, IL; 3Department of Biomedical Engineering, Duke University, Durham, NC. 1

Oral gene therapy aims to treat immune-, gastrointestinal- or systemic diseases by ingestion of genetic materials. Compared to the other routes of administration, the oral route is the most convenient, but also the most challenging for gene delivery. Previous studies have shown that oral delivery of chitosan nanoparticles complexed with 0.6 mg DNA encoding FVIII resulted in phenotypic correction of Hemophilia A. The treated animals, unlike control group survived a tail-clipping test. Polyethyleneimines (PEIs) are among the most commonly studied non-viral vectors for in-vivo applications. They have been evaluated in CNS, lung and intratumoral gene transfer studies. However there is no report on PEI as a carrier for oral gene delivery. We have studied the effects of types of PEI, nature of plasmid DNA, and DNA dose on secreted embryonic alkaline phosphatase (SEAP) expression in mice following oral administration of the SEAP gene. SEAP DNA-PEI nanoparticles having a nitrogen to phosphate (N/P) molar ratio of 10 were warmed with unflavored Gelatin Knox™ and cooled to form cubes. Mice (n=10) were fed with 10g of PEIMolecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

NON-VIRAL GENE DELIVERY DNA nanoparticles as gelatin cubes over a period of two days. The different formulations are shown in Table 1. The effect of the formulation on SEAP expression in mice is seen in Figure 1. The systemic SEAP activity was extremely low in all groups. However, statistically significant activities above background of untreated animals could be observed in the following groups: Formulations A and E on day 2; Formulation C on days 2 and 5; and Formulation D on days 2 and 8. While this study shows that systemic transgene expression can be achieved by oral administration of PEI-DNA nanocomplexes, it also highlights the inefficiency of this delivery system. Work is underway to further optimize the formulation and evaluate other gene carriers. Table 1: PEI-SEAP DNA nanoparticles formulations Group PEI (25 kDa) DNA Plasmid type type dose, mg A Branched 1.25 CMV promoter - human SEAP, gWiz ™ SEAP (Gene Therapy Systems, Inc.) B Linear 1.25 CMV promoter - human SEAP, gWiz ™ SEAP (Gene Therapy Systems, Inc.) C Linear 2.5 CMV promoter - human SEAP, gWiz ™ SEAP (Gene Therapy Systems, Inc.) D Branched 1.25 No CpG motifs - mouse SEAP, pCpGmSEAP (Invivogen) E Linear 1.25 No CpG motifs - mouse SEAP, pCpGmSEAP (Invivogen) F Untreated animals ————-

168. Peptide Aldehyde Inhibitors of the Proteasome as Improved Gene Transfer Agents Molly E. Martin, Ji-Seon Kim, Kevin G. Rice. 1 Medicinal and Natural Products Chemistry, University of Iowa, Iowa City, IA. Proteasomes are multisubunit complexes that are responsible for the degradation of many cytosolic proteins. The barrel-like 20S catalytic core of the enzyme is composed of four heptameric rings that contain the proteolytic sites. The proteasome has multiple peptidase activities that can be classified into three main groups: cleavage after hydrophobic side chains (chymotrypsin-like), cleavage after basic residues (trypsin-like), and cleavage after acidic residues (peptidylglutamyl peptide hydrolysis or PGPH). The proteasome has been shown to be a key route of metabolism for peptide-based non-viral gene delivery systems1. The gene transfer efficiency of peptide-DNA condensates can be enhanced by the addition of commercially available proteasome inhibitors that prevent premature degradation of the gene delivery peptide. MG115 is a tripeptide with a C-terminal aldehyde that binds reversibly to the N-terminal Thr residue of the catalytic subunits thereby inhibiting proteasome activity. When MG115 was administered simultaneously with peptide-DNA condensates, an increase in luciferase expression was observed in both HepG2 and CF/T1 cells, as reported by Kim et al1. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

Previous studies have shown that MG115 can be toxic to cells in a dose dependent manner, with an LD50 of approximately 2 µM. Another difficulty was ensuring the concurrent uptake of both MG115 and peptide-DNA condensates by cells. Here we have described the synthesis and testing of gene delivery peptides containing a C-terminal aldehyde that condense DNA, mediate cellular uptake, inhibit the proteasome, and boost gene transfer efficiency. We also hypothesized that incorporation of the MG115 peptide sequence into a longer peptide could decrease its cytotoxic effects on the cell. The length and sequence of the cationic peptide was varied with attachment of the tripeptide aldehyde at the C-terminus. Incorporation of the peptide inhibitor sequence into the DNA condensate was compared to co-administration of MG115 and peptide-DNA condensates in assays for transfection efficiency and cell viability. 1 Kim, J., Chen, C., and Rice, K. G. “The Proteasome Metabolizes Non-viral Gene Delivery Systems” Gene Therapy (2005) 12, 15811590.

169. Patterned Substrate-Mediated DNA Delivery Using Soft Lithography for Neural Tissue Engineering Tiffany L. Houchin,1 Lonnie D. Shea.1,2 Chemical and Biological Engineering, Northwestern University, Evanston, IL; 2Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL. 1

Combining tissue-engineering scaffolds with gene therapy can provide versatile systems to physically support cellular processes and locally stimulate tissue growth. The engineering of tissues with complex architectures, such as the nervous system, will likely require spatial patterning of molecular signals resulting in concentration gradients of extracellular proteins that organize cells into functional tissues. We believe that substrate-mediated gene delivery, which involves immobilizing DNA complexes to a cell-adhesive substrate, provides the ability to spatially pattern molecular signals that can guide cell migration and organize tissue formation. We have combined substrate-mediated gene delivery with soft lithography to pattern DNA complex deposition and therefore cellular transfection. Non-specific interactions between lipoplexes and a polystyrene surface allow immobilized complexes to remain in the pattern and transfect only cells in the region of the pattern. Polydimethylsiloxane (PDMS) microfluidic devices with channels ranging in width from 100 to 1000 µm were utilized to pattern lipoplexes on the polystyrene surface. Complex binding efficiency in the pattern, as determined using 32P-labeled plasmid, was dependent on PDMS treatment, with no treatment allowing for 15% binding efficiency, and both O2 plasma and Pluronic L35 treated permitting 25% binding efficiency (1000 µm channels). The percentage of transfected HEK293T cells in the pattern was dependent on the PDMS treatment, concentration of DNA in the microchannels, and size of the microchannels. Substantial levels of transfection were observed only with Pluronic treated PDMS microchannels. The percentage of transfected cells ranged from 30% to 40% for the different sized channels with varying DNA concentrations. The capacity of patterned transfection to guide neuron growth cones during axonal extension was investigated using a neuronal coculture system. Primary dorsal root ganglia (DRG) neurons were dissected from 8-day chicken embryos, dissociated, and cultured on patterns of RK5-NGF transfected HEK293T cells. Neurite outgrowth was greater at the location of transfected cells as compared to regions outside the pattern. Additionally, high transfection efficiencies were not necessary to achieve patterns of neurite extension, suggesting that cell types that are more difficult to transfect can be utilized with this system. S65