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IN VIVO APPLICATION all responders occurred in final 2 dose levels, i.e. 4/11 patients. Viral replication confirmed by EM of post-treatment biopsy samples. Quantitative PCR documented CN706 in blood at two different time points, also suggestive of viral replication. Conclusions: CV706, administered by a modified prostate brachytherapy technique, exhibits an acceptable safety profile with both biochemical and histologic evidence of antitumor activity in patients with prostate cancer that is locally-recurrent following radiation. In vivo replication was established in these patients. As we have recently demonstrated substantial in vitro and in vivo synergy when CV706 treatment is followed by radiation, these results support continued clinical development in a Phase I/II study combining treatment with CV706 plus radiation thearpy for initial management of patients with clinically-localized prostate cancer. Supported by NIH S.P.O.R.E. in Prostate Cancer, NIH GCRC Johns Hopkins and a clinical research agreement only with Calydon, Inc.

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875. Widespread Distribution, Uptake and Expression in Rat Brain after Non-viral Delivery of mRNA and DNA to the Lateral Ventricle James G. Hecker, Leon L. Hall, Van R. Irion Transient expression of the inducible, protective protein from the 70 kDa Heat Shock Protein (HSP) gene family, Hsp70-1, by delivery of mRNA or DNA is hypothesized to be a means of improving cellular survival, and would be a novel peri-operative application of gene transfer technology. Upregulation by heat shock of the inducible Heat Shock Proteins is protective against a subsequent, nearlethal stressor, such as ischemia. However, there is currently no practical way to induce endogenous Hsp70, the most protective of the Heat Shock Proteins, in humans. mRNA transfection of vulnerable cells would provide one approach. Transient prophylactic expression of protective intracellular proteins via mRNA or DNA transfection could reduce cell death after a subsequent, severe stressor. We have initiated a series of studies which are designed to demonstrate and quantify the delivery and transient expression of mRNA or DNA for one particular heat shock protein, inducible Hsp70, as a method of protecting the Cental Nervous System (CNS), and as an important illustration of the principle of transient expression. mRNA and DNA transfection and expression of reporter gene proteins and Hsp70 in vivo in rat central nervous system (CNS) and in vitro in neuronal cells using non-viral delivery is described. Cationic lipid/mRNA complexes stabilize the nucleic acid in the extracellular environment. Untranslated stabilizing sequences in the optimized DNA construct, from which mRNA is transcribed, serve the same function in the intracellular milieu. A variety of stabilized mRNA and DNA vectors which encode Hsp70-1, ␤-galactosidase (␤-gal), P. pyralis luciferase, and GFP have been used. Expression of reporter enzymes and Hsp70-1 was previously demonstrated in a variety of neuronal and non-neuronal cells. Transfections were optimized for average luminescence per cell in a cell lysis analysis method, and for maximum percentage of cells transfected using a flow cytometry analysis. In vitro, expression is seen after mRNA delivery in as little as 30 to 45 minutes, with a peak in expression at 6-7 hours. Formulation methods for in vitro transfections were optimized using novel cationic lipids, and these results have been applied in vivo. Nucleic acid/cationic lipid complexes were injected into rat CNS parenchyma or lateral ventricle. Uptake and translation in neurons and neuroglial cells was demonstrated by luciferase chemiluminescence assay, immunohistochemistry, and fluorescence microscopy. Cells which demonstrate the characteristic morphology of pyramidal neurons are clearly identifiable. Initial stereology analysis demonstrates expression in 25,000 to 40,000 neuroglial cells across 30␮ coronal sections and in sections distributed along the longitudinal axis. Results support the further development of mRNA-based Hsp70 therapy for pre-operative MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

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protection from ischemic CNS damage. Supported in part by the Foundation for Anesthesia Education and Research (FAER), the Western States AHA, and NIH/NINDS.

876. Programmable Pharmacokinetics, Disease Site Targeting and Tissue Specific Gene Expression of Stable Plasmid-Lipid Particles for Systemic Gene Delivery and Expression Ian MacLachlan*, David Fenske†, Ellen Ambegia*, Lorne Palmer*, Mark Murray*, Pieter Cullis† *Protiva Biotherapeutics Inc., Burnaby, B.C. †Liposome Research Group, University of British Columbia, Vancouver, B.C. The SPLP method results in the encapsulation of plasmid DNA in small (diameter⬃70 nm) “stabilized plasmid-lipid particles” (SPLP). SPLP consist of one plasmid per particle, encapsulated within a lipid bilayer stabilized by the presence of a poly(ethyleneglycol) (PEG) coating. SPLP exhibit extended circulation lifetimes following intravenous administration and promote delivery of intact plasmid to distal tumor sites resulting in reporter gene expression at the disease site. Here we describe in detail the disease site targeting and gene expression resulting from intravenous administration of SPLP in tumor bearing mice. SPLP with long circulation times accumulate to levels corresponding to five to ten percent of the total injected dose per gram of tumor or greater than 1000 copies of plasmid DNA per cell, giving rise to levels of gene expression that are more than two orders of magnitude greater than those observed in any other tissue. Interestingly, although the liver accumulates 20-30% of the total injected dose, very low levels of gene expression are observed in the liver. This is thought to be due to the limited hepatocellular uptake of the PEG-ylated SPLP. The cellular uptake and gene expression mediated by PEG-lipid containing transfection reagents remains low when compared to non-PEG-ylated systems. Here we show that the in vivo transfection potential of PEG-lipid containing systems can be enhanced through the incorporation of a cationic PEG lipid (CPL) consisting of a DSPE anchor, PEG3400 spacer chain and a cationic head group. When CPL are incorporated into SPLP at concentrations of 2 to 4 mol% the resulting CPL-SPLP are of a similar size and stability as native SPLP. Incorporation of CPL results in a dramatic increase in intracellular delivery and a concomitant increase in transfection activity measured both in vitro and in vivo. Specifically, CPL-SPLP yielded 105 fold more in vitro gene expression than native SPLP. When CPL-SPLP are administered intravenously they yield a substantial (250 fold) increase in hepatic gene expression compared to native SPLP. The increase in CPL-SPLP potency is specific to the liver. The levels of gene expression measured in the lung, kidney, spleen or heart remain unchanged, contributing to more than two orders of magnitude differential in the gene expression measured in the liver vs. other organs. These results illustrate the potential for modulating the transfection properties of PEG-lipid containing systems while retaining the stability and small uniform size required to achieve systemic gene delivery. In particular they demonstrate that disease site targeting and tissue specific gene expression can be re-programmed by altering the lipid composition of non-viral gene delivery systems.

877. PET-BASED IMAGING OF HSV-1-tk GENE EXPRESSION IN A PHASE I/II CLINICAL GLIOMA GENE THERAPY TRIAL A. Jacobs*†, J. Voges‡, R. Reszka¶, M. Lercher*, A. Gossmann§, L. Kracht*, C. Kaestle†, N. Weisner‡, R. Wagner*, K. Wienhard*, V. Sturm‡, W.D. Heiss*† *MPI for Neurological Research, Cologne, Germany †Department of Neurology, University of Cologne, Germany ‡Department of Stereotactic & Functional Neurosurgery, University of Cologne, Germany §Department of Radiology, University of Cologne, Germany ¶ Max-Dellbru¨ck-Center of Molecular Medicine, Berlin, Germany Objective: To non-invasively localize the magnitude of liposomal vector-mediated herpes simplex virus type 1 thymidine kinase

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(HSV-1-tk) gene expression in patients with a recurrent glioblastoma participating in a gene therapy trial investigating the safety of intratumorally infused liposome-gene-complex [LGC] followed by ganciclovir [GCV]. Methods: Five patients (age: 49-67) received a stereotactically guided intratumoral infusion (max. flow: 1.5 ml/h, volume 30 ml) of LGC (DAC-Chol/DOPE [w:w; 70:30], HSV-1-tk bearing plasmid; 1). Two days later, positron emission tomography (PET) was performed post injection (p.i.) of 1.6-4.0 mCi I-124-labeled 2⬘-fluoro-2⬘-deoxy-1-ß-D-arabinofuranosyl-5-iodo-uracil ([I-124]FIAU), a specific marker substrate for HSV-1-TK expression (2-4). To study the kinetics of tracer accumulation and wash-out, a series of emission-scans were acquired until 68 hours p.i.. An FIAU-PET before LGC application served as baseline. GCV treatment (2 x 5 mg/kg/bw; 14 days) was carried out starting four days after LGC infusion. Treatment response was recorded by MRI, [F-18]-FDG- and [C-11]-MET-PET. Results: In one out of five patients specific [I-124]-FIAU-related radioactivity was observed within the infused tumor. The volume of specific FIAU-related radioactivity demonstrated (i) a significant increase of the accumulation rate, Ki, of [I-124]-FIAU from 0.047 at baseline to 0.096 ␮l/min/g after vector administration (two-compartment model); (ii) an increase of the tissue/plasma radioactivity ratios over time (Patlak-analysis), both indicators for specific radiotracer trapping and, hence, HSV-1-TK expression; and (iii) the signs of necrosis after GCV treatment (FDG-/ MET-PET). In four patients no specific FIAU-accumulation was observed. Conclusions: These results demonstrate for the first time that FIAU-PET imaging of vector mediated HSV-1-TK expression in the clinical setting is feasible and that vector-mediated gene expression can be correlated to the therapeutic effect. This method will contribute to the development of standardized gene therapy protocols and of efficient and safe vector applications in humans. References: (1) Zhu J et al. Gene Therapy 1996;3:472-476. (2) Tjuvajev JG et al. Cancer Research 1995;55:6126-6132. (3) Tjuvajev JG et al. Cancer Research 1996;56:4087-4095. (4) Tjuvajev JG et al. Cancer Research 1998;58:4333-4341. Supported in part by BMBF grant 0311111.

878. Non-invasive CNS Gene Delivery by Retrograde Transport of Polymeric and Liposomal DNA Complexes Shu Wang*, Nan Ma*, Shujun Gao*, Hanry Yu*, Kam W. Leong*† *Biomaterials/Tissue Engineering Program, Institute of Materials Research & Engineering, National University of Singapore, 3 Research Link, Singapore 117602, †Department of Biomedical Engineering, Johns Hopkins University, Baltimore, USA Nonviral chemical-based gene delivery systems have been developed to avoid potential problems inherent in viral gene vectors. As neurons are capable of uptaking exogenous macromolecules from the muscles that they innervate, we investigated the feasibility of achieving gene transfer in brainstem neurons by peripheral intramuscular injection of plasmid DNA complexed with the cationic polymer, polyethylenimine (PEI) in the rat hypoglossal system. Using the luciferase reporter gene driven by a Rous sarcoma virus promoter, a high level of transgene expression of up to 4 x 106 RLU per brainstem at 20 mg of plasmid DNA was achieved after tongue injection. This transfection efficiency is close to what is achievable by direct brain injection. Control injections with PEI solution or DNA alone did not yield any luciferase activities in the brainstem. Using lacZ as a reporter gene, transgene expression in the brainstem was localized in hypoglossal motor neurons, a group of neurons that innervate tongue muscles. PCR analysis of brainstem samples detected the plasmid DNA injected into the tongue, demonstrating that the PEI/DNA complexes had migrated by retrograde axonal transport to neuronal cell bodies in the brainstem after being internalized by nerve terminals in the tongue muscle. Using a therapeutic

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NAKED DNA GENE TRANSFER Bcl-2 gene driven by a cytomegalovirus promoter and western blotting, transgene expression was detectable in the brainstem as early as 18 hours after tongue injection and lasted for at least 2 weeks. Two lipid transfection agents GenePORTERTM and TransFastTM mediated a weak gene expression in the hypoglossal system, but not two polymers, poly-L-lysine and chitosan. Bcl-2 expression in the spinal cord was also achieved after injection of PEI/DNA complexes into the gastrocnemius muscle. Gene delivery into the brain without tissue destruction is challenging. The neuronal gene delivery method established in this study bypasses the blood-brain barrier and suggests a possible therapeutic strategy for safe and efficient CNS gene transfer. By choosing appropriate muscles for injection, it is possible to target many brain areas and spinal cord regions for experimental gene manipulation or clinical gene therapy.

879. Sequential Injection of Cationic Liposome and Plasmid DNA Delivers Gene Effectively to the Lung with Minimal Inflammatory Toxicity Yadi Tan*†, Feng Liu*, Zhiyu Li*, Leaf Huang*† *Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh †Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213 Application of lipoplex based gene vector has been hurdled by the acute inflammatory toxicity associated with its systemic administration. In the current study, a safe, simple and effective alternative to lipoplex, i.e., sequential injection of cationic liposome and plasmid DNA, was evaluated. When plasmid DNA was injected into the tail vein of mice 2-5 min after the injection of cationic liposome, there was a 70-90% reduced induction of pro-inflammatory cytokines, including TNF-␣, IL-12 and IFN-␥, compared with that from lipoplex injection. In addition, mice received sequential injection yielded a 2-5 fold higher level of transgene expression in the lung, and exhibited shorter refractory period for repeated dosing than those received lipoplex. Furthermore, other types of toxicity associated with lipoplex, such as neutropenia, lymphopenia, platelet and complement depletions, were also significantly reduced with sequential injection. Such reduction in cytokine induction was observed with several different liposome formulations and appeared to be a general phenomenon. Research supported by NIH grant AI48851.

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880. Angiogenic Gene Therapy for the Induction of Coronary Collateral Formation in a Porcine Model of Chronic Myocardial Ischemia: A Randomized, Blinded, Placebo-Controlled Study Michael Kutryk, Reena Sandhu, Andrew Campbell, Gary Salasidis, Yves Leclerc, Assim Cheema, David Fitchett, Duncan Stewart Terrence Donnelly Heart Centre, St Michaels Hospital, Toronto Background: Intramyocardial injection of plasmid DNA incorporating the VEGF transgene has been shown to reduce symptoms of myocardial ischemia. Angiopoietin-1 (Ang-1) is a newly described angiogenic factor that has been shown developmentally to induce the formation of larger, more complex vascular networks. However, the effects of Ang-1 on coronary collateral formation are not known. The aim of this study was to investigate the effects of direct intra-myocardial injection of plasmid DNA incorporating the Ang-1 transgene, either alone or with VEGF, on myocardial collateral blood flow and infarct size in pigs. Methods: Through a left lateral thoracotomy, juvenile Yorkshire swine (25-30 kg) were fitted with an ameroid constrictor MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy