Site-Specific Genomic Integration in Hematopoietic Cells

NAKED DNA-BASED GENE TRANSFER received virus under the conditions to be tested (different volume, vehicle, number of injections, etc). One day after viral delivery, dogs were administered a clinically-relevant dose of radioisotope and after 1 hour of uptake, excised prostates were frozen and sectioned for autoradiography. The technique allowed for head-to-head comparisons of conditions that are thought to improve adenoviraldelivered gene expression. Our results predict that more than 150 viral deposits (100 ml each) are needed for complete prostate coverage using a simple linear extrapolation of spatial distribution from a single viral injection when the vehicle is saline. The number of viral deposits needed to cover the organ drops to between 40 and 80 when virus was suspended in PEG 400 instead of saline. We have begun testing the prediction experimentally and have found that adenovirus administered in four small volume deposits (100 ml) provided better gene coverage than adenovirus deposited in a single large deposit (1000 ml). The methodology presented provides the framework for optimized adenoviral delivered gene therapy.

1035. Syncytia Induction Enhances the Oncolytic Potential of Vesicular Stomatitis Virus in Virotherapy for Cancer Oliver Ebert,1 Katsunori Shinozaki,1 Chryssanthi Kournioti,1 Man-Seong Park,2 Adolfo Garcia-Sastre,2 Savio L. C. Woo.1 1 Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY; 2Department of Microbiology, Mount Sinai School of Medicine, New York, NY. Vesicular stomatitis virus (VSV) selectively replicates in tumor but not in normal cells, and is being developed as an oncolytic agent for cancer therapy. Here we report the construction of a recombinant VSV capable of inducing syncytia formation between tumor cells through membrane fusion at neutral pH, which led to enhanced oncolytic properties against multi-focal hepatocellular carcinoma (HCC) in the livers of immune-competent rats. Recombinant VSV vectors were constructed by insertion into its genome a transcription unit expressing a control or fusion protein derived from Newcastle disease virus. In vitro characterization of the recombinant fusogenic VSV vector on human and rat HCC cells showed extensive syncytia formation and significantly enhanced cytotoxic effects. In vivo, administration of fusogenic VSV into the hepatic artery of Buffalo rats bearing syngeneic multi-focal HCC lesions in their livers resulted in syncytia formation exclusively within the tumors, as well as no collateral damage to the neighboring hepatic parenchyma. The fusogenic VSV also conferred a significant survival advantage over a non-fusogenic control virus in the treated animals (p=0.0078, logrank test). The results suggest that fusogenic VSV can be developed into an effective and safe therapeutic agent for cancer treatment in patients, including those with multi-focal HCC in the liver.

NAKED DNA-BASED GENE TRANSFER 1036. Effective Acceleration of Wound Healing by Simultaneous Transfestion of Hepatocyte Growth Factor Gene and Prostacyclin Synthase Gene Using Shima Jet Yasuo Kunugiza,1 Naruya Tomita,2 Tetsuya Tomita,1 Hideo Hashimoto,1 Keita Yamasaki,2 Hiromi Koike,2 Hideki Yoshikawa,1 Ryuichi Morishita.2 1 Orthopaedic Surgery, Osaka university, Suita City, Osaka, Japan; 2Clinical Gene Therapy, Osaka university, Suita City, Osaka, Japan. Background: Gene delivery into skin may be useful for treating skin diseases. We tried to evaluate the effectiveness of intradermal injection of naked plasmid into the skin by spring powered jet Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

injector, Shima Jet, that was originally developed as non-needle jet injector of insulin for diabetic patients. Recently, there have been a lot of interests in the role of Hepatocyte Growth Factor (HGF) for wound healing. Re-epithelialization and granulation formation induced by HGF would promote the wound closure. On the other hand, the role of Prostagrandin I2 (Prostacyclin) in the vasolidation of blood vessels has been focused on and the effctiveness of Prostagrandin I2 Synthetase (PGIS) gene therapy for promoting blood flow has been reported. In this study we evaluated the effectiveness of gene therapy for healing of impaired wound by transfecting these genes together to the skin by Shima Jet. Material and methods: We transfected luciferase gene in the back of rat by Shima Jet and, 24 hours later cut off the skin and examined the local expression of luciferase. Next, we transfected lacZ gene in the back of rat by Shima Jet and 24 hours later examined the local expression area of gene by beta-galactosidase staining. Moreover, we made full thickness wound (1.6 cm in diameter) in the back of steroid-induced healing impaired rats and on the same day transfected HGF gene (100 mg, 5 points) and/or PGIS gene (100 mg, 5 points) around the wound by Shima Jet. We measured the local expresion of HGF protein by ELISA (day 2) and blood flow around the wound by Laser Doppler imaging (day 4) and the wound area (days 0, 2, 4, 6, 9, 11, and 16). Results: The local expression of luciferase was about 100 times higher when transfected by Shima Jet than that by needle transfection. Gene expression of luciferase was observed in epidermis around the injection sites. The higher expression of HGF protein (HGF: 11.5 pg/mg tissue, control: 0.4 pg/mg tissue, p<0.01) was observed in HGF gene transfected group compared to the control group. Compared to control group, significant increase of blood flow was observed in PGIS gene transfected group (p<0.05). Significant promotion of wound closure was observed in PGIS or HGF gene transfected groups, and further promotion was observed in both HGF and PGIS gene transfected group (days 4 and 6) (p<0.05) . Conclusion: Gene transfer by Shima Jet would be a new method for gene delivery to the skin. Co-transfection of HGF and PGIS gene using Shima Jet would be an effective strategy to wound healing.

1037. Site-Specific Genomic Integration in Hematopoietic Cells Roger P. Hollis,1 Sarah Nightingale,1 Michele P. Calos,2 Gay M. Crooks,1 Donald B. Kohn.1 1 Department of Research Immunology / BMT, Childrens Hospital Los Angeles, Los Angeles, CA; 2Department of Genetics, Stanford University, Stanford, CA. Methods of gene transfer to hematopoietic stem cells (HSCs) that result in safe stable chromosomal integration and persistent gene expression in vivo are desired. HSC are an attractive target as their modification could provide treatments for many inherited blood diseases such as Severe Combined Immunodeficiency (SCID) and acquired diseases such as infection by Human Immunodeficiency Virus (HIV-1). PhiC31 integrase is a site-specific recombinase that catalyses a specific, unidirectional integration reaction between two distinct DNA sites known as attB and attP. It has been demonstrated previously that a plasmid expressing the integrase can mediate the integration of a co-delivered attB-containing plasmid into mammalian chromosomes at pseudo attP-sites (host sites sharing homology to attP, as recognized by phiC31). Here we delivered the non-viral phiC31 integrase system to achieve site-restricted integration of genes into the genome of hematopoietic cells using the AMAXA nucleoporator. We applied the phiC31system to integrate a MNDdriven eGFP reporter gene into the genomes of human hematopoietic cell lines and primary CD34+ progenitor cells. In the K562 cell line, we observed stable expression of eGFP in >60% of cells for over 2 S397

NAKED DNA-BASED GENE TRANSFER months; in contrast, in control cells not treated with integrase, the level of gene expression had fallen to near background (<1 %) by 2 weeks. In purified human cord blood and bone marrow CD34+ progenitor cells, the integrase repeatedly improved marking efficiency when compared to controls. However, to date relatively low levels of cells that express eGFP (1% range) have been observed over 4-6 weeks of long-term culture. A LAM-PCR based method was applied to investigate pseudo attP-sites in the marked hematopoietic cells. Analyses of integration sites in these marked cells indicated that the preferred pseudo attP-sites chosen by the phiC31 integrase in hematopoietic cells may differ from those previously reported in other commonly used human cell lines and primary cells. Integration events into these pseudo attP-sites are directly responsible for the high level of marking seen in these hematopoietic studies. We are currently defining the identity and expression context afforded by hematopoietic-preferred integration sites. These encouraging results of long-term stable expression highlight the potential of this likely safer non-viral method for ameliorating diseases affecting the hematopoietic system.

1038. Intramuscular Plasmid DNA Electrotransfer. Biodistribution, Degradation and Stability of the Transfectable Pool of Plasmid DNA Michel-Francis Bureau,1 Naimi Sadoui,2 Johanne Seguin,1 Daniel Scherman.1 1 Laboratoire de Pharmacologie Chimique et Génétique INSERM U640-CNRS FRE2463, Paris, France; 2Aventis Pharma-Gencell S.A., Vitry sur Seine France, France. In the present work, we used plasmid DNA radiolabelled with 3H by methylation (10) to study radiolabelled plasmid DNA biodistribution and degradation in the muscle at different times after injection, with or without electrotransfer using previously defined conditions. Radiolabelled plasmid progressively left the muscle and was degraded as soon as 5 minutes after plasmid injection, with or without electrotransfer. Autoradiography showed that the major part of injected radioactivity was detected in the interfibrilar space of a large proportion of the muscle. Large zones of accumulation of radioactivity which seems to be into some fibres (more than 20 µm) were identified as soon as 5 minutes after electrotransfer. Such structures were never observed on slices of non-electrotransfered muscles. However, these structures were not frequent and probably lesional. The surprising fact is that despite the amount of intact plasmid having been greatly reduced between 5 minutes and 3 hours after injection, the level of transfection remains unchanged whether electric pulses were delivered 20 sec, 1 hour, 2 hours or 3 hours after injection. Such a behavior was similarly observed when injecting 0.3, 3 or 30 µg of plasmid DNA. Moreover, the transfection level was correlated to the amount of plasmid DNA injected. These results suggest that as soon as it is injected, plasmid DNA is proportionally partitioned between at least two compartments. While a major part of plasmid DNA is rapidly cleared and degraded, the electrotransferable pool of plasmid DNA represents a very small part of the amount injected and belongs to another compartment where it is protected from endogenous DNAses.

1039. Mechanistic Insights into the Persistence of Non-Viral Mediated Gene Transfer In Vivo Efren Riu,1 Zan Huang,1 Mark A. Kay.1 1 Pediatrics and Genetics, Stanford University, Stanford, CA. Non-viral vectors offer great potential for clinical application in gene therapy trials. These vectors have several advantages over the viral-based gene therapy approaches, including non-toxicity and ease of production. However, the ability to achieve reproducible, persistent, and therapeutic levels of gene expression in vivo by nonS398

viral gene transfer approaches has been problematic because of the relatively transient nature of transgene expression in vivo. This may limit the use of these vectors in the clinic. Understanding the mechanisms required for persistent gene expression will be helpful for vector design and eventual clinical implementation. We have suggested that covalent linkage of bacterial DNA to the expression cassette plays a critical role in transcriptional silencing of the transgenes in vivo. To gain an insight into the role of the covalent linkage of plasmid DNA to the expression cassette and transcriptional repression, and whether this silencing effect could be alleviated by altering the molecular structure of vector DNAs in vivo, we generated a scheme for converting routine plasmids into a purified expression cassette, free of bacterial DNA after gene transfer in vivo. To do this, we co-injected the I-SCeI cDNA together with a plasmid reporter containing either the human alpha-1-antitrypsin (hAAT) or the human clotting factor IX (hFIX) expression cassette flanked by two I-SCeI sites. Two weeks after DNA administration, mice injected with the reporter gene alone or with the irrelevant control plasmid showed very low serum levels of hAAT and hFIX, which remained low throughout the length of the experiment. However, animals that expressed I-SCeI had a 5-10 fold increase in serum hAAT and hFIX that persisted for at least 8 months (length of study). In addition, expression of I-SCeI resulted in cleavage and excision of the expression cassettes from plasmid backbone, forming mostly circles devoid of bacterial DNA sequences, as established by a battery of different Southern blot and PCR analyses (in vivo and in vitro) in both C57Bl6 and scid mice. In contrast, only the input parental circular plasmid DNA band was detected in mice injected either with the reporter gene alone or an I-SCeI plasmid with the hAAT reporter plasmid lacking the I-SCeI sites. This study presents further independent evidence that removing the covalent linkage between plasmid and transgene sequences leads to a marked increase and persistence of transgene expression. Thus, unraveling the mechanisms by which the covalent linkage of bacterial DNA to the expression cassette is connected to gene silencing is fundamental to establish the mechanism of transcriptional regulation in mammalian systems and will be important for the development of versatile non-viral vectors that can be used to achieve persistent gene expression in different cell types.

1040. A Direct Comparison of Two Non-Viral Gene Therapy Vectors for Somatic Integration: In Vivo Evaluation of the Bacteriophage Integrase PhiC31 and the Sleeping Beauty Transposase Anja Ehrhardt,1 Hui Xu,1 Zan Huang,1 Mark A. Kay.1 1 Department of Pediatrics, School of Medicine, Stanford University, Palo Alto, CA. Gene therapy approaches for genetic disorders require sustained transgene expression levels. Various non-viral vector systems utilizing different recombinases for somatic integration were developed and tested in vivo. In this study we performed a head to head comparison of the site-specific integrase phiC31 derived from a Streptomyces phage and the Sleeping Beauty (SB) tranposase, a member of the TC1/mariner superfamily of transposable elements. For a direct in vivo comparison we tested one of our most robust human coagulation FIX (hFIX) expression cassettes, in which a hFIX minigene is driven by the liver specific human-alpha1antitrypsin promoter and liver specific enhancers. C57Bl/6 mice were either co-injected with a vector expressing the phage integrase phiC31 and a second vector containing the hFIX expression cassette with the recombinase recognition site attB (n=10, phiC31 group) or a SB transposase expressing vector and the corresponding transposon with the identical hFIX expression cassette (n=10, SB group). Mouse livers of control groups (n=10 per group) were infused with vectors expressing a mutant phage integrase or an inactive version of the SB Molecular Therapy Volume 9, Supplement 1, May 2004

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