Physical Method Based Gene Therapy

PHYSICAL METHOD BASED GENE THERAPY in vivo imaging of therapeutic gene expression in tumor tissue infected with any recombinant HSV-1 vector, where a therapeutic gene is substituted for the lacZ gene. References: (1) Tjuvajev JG et al. Cancer Research 1995;55:6126-6132. (2) Tjuvajev JG et al. Cancer Research 1996;56:4087-4095. (3) Tjuvajev JG et al. Cancer Research 1998;58:4333-4341. (4) Jacobs A et al. J Cereb Blood Flow Metab 1999;19:S294. Supported in part by ZMMK grant TV46 and MSWWF grant 516-400 002 99.

PHYSICAL METHOD BASED GENE THERAPY 158. Gene Delivery of Calcitonin Gene-related Peptide in Animal Models Lichun Sun*†, Jiong Tang*‡, Zhaorui Ren*, Xinhui Ge*, Guangjing Li*, Hanzhang Ye*, Zhaoxin Zheng* *School of Life Science, Fudan University, Shanghai 200433, China †Department of Medicine, Tulane University Medical School, New Orleans, LA 70112, USA ‡Developmental, Cellular and Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Texas, USA Calcitonin Gene-Related Peptide (CGRP) is a promising therapeutic agent in cardiovacular diseases. CGRP gene with a short signal peptide was synthesized and linked together, then cloned into two different eukaryotic expression vectors pBPV and pCDM8. Both two recombinant vectors pBPV-CGRP and pCDM8-CGRP carrying CGRP gene were respectively delivered into the animal models: Spontaneously Hypertentive Rats (SHR) and Artificially Hypertensive Beagles (AHB). These recombinant vectors were proved to express CGRP in both animal models. The results showed they greatly decreased blood pressure of the animal model (even up to 24.42% with pBPV-CGRP, 20.75% with pCDM8-CGRP in SHR group ; over 10% with pCDM8-CGRP in AHB group compared to control) and increased CGRP concentration (up to maximum value: 132.00% with pBPV-CGRP, 102.40% with pCDM8-CGRP in SHR group; 79.85% with pCDM8-CGRP in AHB group compared to control) in blood. The CGRP concentration in blood came to increase as the blood pressure decreased, and then went down as blood pressure went up, Higher DNA dose also had more obvious effects on SHRs. The maximum value of SHR’s blood pressure was respectively 20.75% in 500ug, 16.00% in 200ug and 13.46% in 100ug of pCDM8CGRP DNA each SHR. And the effects could be remained for almost two months. CGRP expression was also supported by RT-PCR and Immunohistochemistry after DNA injection. So our study suggested that CGRP gene delivery could decrease blood pressure and might be a possible candidate way for treating high blood pressure.

159. Local Gene Transfer into Rat Kidney Using Low Voltage Ultrasound with Contrast Microbubbles: Development of Novel Non-Viral Gene Transfer Hiromi Koike*, Ryuichi Morishita*, Naruya Tomita†, Yoshiaki Taniyama*, Katsuro Tachibana‡, Yasufumi Kaneda* *Division of Gene Therapy Science, Osaka University Medical School †Department of Geriatric Medicine, Osaka University Medical School ‡First Department of Anatomy, Fukuoka University School of Medicine Safety issue of gene therapy is quite important for human gene therapy. From this viewpoint, it is necessary to develop a novel non-viral efficient gene transfer method. Recently, it is reported that therapeutic ultrasound can induce cell membrane permeabilization. In this study, we transfected luciferase plasmid to the rat kidney by using low voltage therapeutic ultrasound with contrast microbubbles and measured luchiferase activity in transfected kidney, and performed biochemical blood test (Crea, Urea-N, GOT, GPT). We transfected luciferase plasmid mixed

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with microbubbles into the left kidney of rat and treated with ultrasound for 2 minutes. Three days after treatment, activity of luciferase in kidney treated with microbubbles and ultrasound significantly increased in a dose-dependent manner as compared to the control. We used 50 ug luciferase mixed with microbubbles at five different concentrations (concentration: 5%, 10%, 25%, 50%, and 100%). Microbubbles and the therapeutic ultrasound enhanced luciferase activity compared to the control (control ⫽1, 5% ⫽ 6.69, 10% ⫽ 12, 25% ⫽ 122, 50% ⫽ 312, 100% ⫽ 600 P⬍0.01). Moreover, treatment with microbubbles and ultrasound did not influence on chemical blood data. Overall, ultrasound transfection method with microbubbles enhanced transfection efficiency of plasmid DNA into the kidney. This novel method may be useful for clinical gene therapy for renal disease without viral vector system.

160. Generation of Transcriptionally Active PCR Fragments with TAP-Express™ Xiaowu Liang, Andy Teng, Dawn Braun, Jiin Felgner, Philip Felgner Gene Therapy Systems, Inc., San Diego, CA Access to complete genome sequence information for an evergrowing list of organisms has ushered in a new era in biology and biotechnology. Where we once sought genes for functions, we now increasingly seek functions for genes. The limiting factor in many “functional genomics” strategies is the sheer volume of manipulations needed to systematically screen large numbers of gene products for a particular activity, and this has led to the introduction of robotics and other high throughput technologies into the biology laboratory. The advent of PCR freed us from the need to rely on microorganisms to produce useable quantities of DNA, but until now, the only way to create transcriptionally active genes was to clone PCR fragments into an expression vector, transform bacteria, and purify the plasmid. Although this process can yield large quantities of plasmid, it is time- and labor-intensive, particularly when more and more genes are simultaneously examined. It would greatly accelerate genomics research and functional screening if the initial PCR product were transcriptionally active. In response to this unmet need, we have produced a kit that allows investigators to convert any gene-of-interest into a Transcriptionally Active PCR (TAP) fragment that is ready for transfection into cells or injection into animals. Because of its simplicity and speed, the TAP Express™ technology is a powerful tool for functional genomics and basic biological research. Besides offering time- and cost-savings benefits, the TAP Express kit allows large numbers of gene products to be screened systematically in functional assays. The TAP Express™ kit is the first and only tool that considerably speeds up the process from gene selection to protein expression by eliminating previously required molecular cloning and plasmid purification manipulations and materials. With TAP Express technology, the gene-ofinterest is ready for expression in one day. The technology uses nested PCR, in which two or more DNA fragments can be joined in a desired orientation, to introduce functional promoter and terminator sequences onto the gene-of-interest. The mixture of overlapping sequences that is included in the TAP Express kit makes this process fast, easy, and reliable. The entire process consists of only two PCR steps. After these two reactions are completed, the PCR product can be used directly in in vitro transfection assays or in vivo. TAP fragments encoding green fluorescent protein (GFP), chloramphenocol aceyl transferase (CAT) and hepatitis-B surface (HBs) antigen were amplified. In each case the PCR amplified product contained the promoter and terminator sequences flanking the coding sequence and each product gave a single band on agarose gels. TAP fragments encoding these reporter genes fragments and transfected into COS 7 and CHO K1 cells expressed transgene at levels comparable to supercoiled pcDNA 3.1 plasmid. Following intramuscular injection into mice, the naked TAP-CAT fragment expressed CAT levels comparable to the MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

PHYSICAL METHOD BASED GENE THERAPY pcDNA 3.1-CAT vector. Similarly, the TAP-HBs fragment injected into mice produced HBs antibody titers comparable to the pcDNA 3.1-HBs vector. These results show that TAP Expression fragments provide a convenient and effective alternative to conventional cloning.

161.

Efficient DNA electrotransfer into tumors

Daniel SCHERMAN, Micka¨el BETTAN, Marie-Agne`s IVANOV, Luis M. MIR, F. BOISSIERE, Pia DELAERE DNA transfer of antiproliferative genes or of genes coding for immuno-modulatory or antiangiogenic products to tumor cells is a promising approach for cancer therapy. However, intratumoral injection of plasmid DNA either “naked” or associated to chemical vectors results in low level of gene expression. Recently, electrically mediated gene transfer has been described to strongly increase foreign gene expression in various tissues. We confirm and extend these observations using a reporter gene encoding for luciferase and long duration electric pulses applied to several murine and human tumor models. After plasmid intratumoral injection, 8 electric pulses of 20 ms duration were delivered at a frequency of 1 Hertz through 2 flat parallel stainless steel electrodes placed at each side of the tumor. Optimal gene transfer was obtained using a voltage to distance ratio comprised between 400 and 600 V/cm. Two days after DNA electrotransfer, we obtained a 10 to 1200 fold increase in gene expression over the naked DNA injection alone, leading to the expression of 0.6 to 300 ng luciferase per tumor. Moreover, histological results using b-Gal reporter gene injected in H1299 tumors indicate that electrotransfer leads to a substantial increase in the percentage of b-Gal positive cells. These results confirm the wide potential of electrotransfer for gene therapy in cancer.

162. Intramuscular DNA electrotransfer. Importance of association between muscle fibers permeabilization and DNA electrophoresis to obtain a high transfection level Daniel Scherman, Michel F. BUREAU, J. GEHL, Virginie DELEUZE, Luis M. MIR Gene transfer using electrical pulses is a rapidly expanding field. Many studies have been performed in vitro to elucidate the mechanism of DNA electrotransfer. In vivo, the use of efficient procedures for DNA electrotransfer in tissues is recent, and the question of the implied mechanisms is largely open. One of the major, well-known effects of the electric field on biological membranes is their permabilization. However contrary to the electropermeabilization induced uptake of small molecules into muscle fibers, plasmid DNA has to be present in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer which could be electrophoresis. In this study we have evaluated the effects of various combinations of square-wave electric pulses of variable field strength and duration, on cell permeabilization and on DNA transfection in the skeletal muscle in vivo. One high voltage pulse of 800 V/cm, 0.1 ms duration (short high pulses) or a series of 4 low voltage pulses of 80 V/cm, 83 ms duration (long low pulses) slightly amplified transfection efficacy, while no significant permeabilization was detected using the 51Cr-EDTA uptake test. By contrast, the combination of one short high pulse followed by 4 long low pulses led to optimal gene transfer efficiency, while inducing muscle fibers permeabilization. These results are consistent with additive effects of electropermeabilization and DNA electrophoresis on electrotransfer efficiency. Finally, the described new combination, as compared to the previously reported use of repeated identical pulses of intermediate voltage, leads to similar gene transfer efficiency, while causing less permeabilization and thus being likely less deleterious. Thus, combination of pulses of various strengths and durations is a new procedure for skeletal muscle gene transfer that may MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

represents a clear improvement in view of further clinical development.

163. Uptake by mouse liver and intracellular fate of plasmid DNA after a rapid tail vein injection of a large volume Simone Wattiaux-De Coninck, Miche`le Lecocq, Robert Wattiaux, Michel Jadot Laboratoire de Chimie Physiologique, Faculte´s Universitaires Notre Dame de la Paix, 5000 Namur, BELGIUM Rapid tail vein injection of a large volume of plasmid DNA solution into mouse enables foreign gene expression in liver (Liu et al. 1999, Gene Ther. 6, 1256-1266; Zhang et al. 1999, Hum. Gene Ther. 10, 1735-1737). The mechanism of that phenomenom is not clearly understood. We have investigated the problem by following the uptake by the liver and the intracellular fate of plasmid DNA labelled by nick translation with 35S dATP. Plasmid DNA was dissolved in 0.2 ml or 2 ml of saline and injected in the tail vein in a few seconds. In both cases, blood radioactivity decreases very quickly and radioactivity accumulates in the liver during the first 10 min. After a 2 ml injection, liver radioactivity remains constant during at least 1h and is mainly acid precipitable. On the contrary, after a 0.2 ml injection, liver radioactivity decreases relatively rapidly and becomes largely acid soluble. Intracellular distribution of radioactivity was investigated by centrifugation methods. After a 2ml injection, a high proportion of radioactivity, mainly acid precipitable is recovered in a fraction isolated at low speed centrifugation (N fraction of de Duve et al. 1955, Biochem. J. 60,604-617). Isopycnic centrifugation in a sucrose gradient indicates that these structures exhibit a median equilibrium density of about 1.20 g/ml. Radioactivity originating from a 0.2 ml injection is mostly found in the cytoplasmic extract where it is shared between the mitochondrial fractions and the soluble fraction. Isopycnic centrifugation shows that radioactivity associated with the mitochondrial fractions is probably located in lysosomes. In summary, our investigations, show that after a rapid tail vein injection of a large volume, a high amount of plasmid DNA remains associated in a liver compartment for a long time without being degraded. Such compartment could be used as a reservoir of DNA for hepatocytes, allowing these cells to be supplied with these molecules and to remain transfected for a long time.

164. High and sustained blood secretion of human alkaline phosphatase and factor IX after electric pulsemediated gene transfer into skeletal muscle Daniel Scherman, Micka¨el BETTAN, Florence EMMANUEL, Raphae¨l DARTEIL, Jean-Michel CAILLAUD, Fabienne SOUBRIER, Pia DELAERE, Didier BRANELEC, Abderrahim MAHFOUDI, Nicolas DUVERGER Numerous diseases linked to the absence or insufficient concentration of a specific plasma protein. Gene transfer is an appealing strategy for correction of such diseases. We report high and sustained plasma secretion of human secreted alkaline phosphatase and of human factor IX by skeletal muscle of mice. This was obtained by delivering square-wave unipolar electric pulses of low field strength (200 V/cm) and long duration (20 ms) to skeletal muscle previously injected with plasmid DNA encoding for the secreted protein. This intramuscular electrotransfer method allows 30 to 150 fold increase in reporter protein secretion, as compared to simple plasmid DNA injection. This increase allows to obtain values of up to 2200 ng/ml of a reporter circulating protein. Moreover, this high level of secretion remains stable for twelve months.

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PHYSICAL METHOD BASED GENE THERAPY 165. Simple method of extraction and purification for high purity, large scale, and large quantities of plasmid DNA AKIHIKO ISHIDA*†, Keishi Sekiguchi*, Fumioki Yasuzumi*, Sakae Arakaki‡, Masaru Nagamine§ *Second Department of †Anatomy, School of Medicine, University of the Ryukyus ‡Advanced Medical Biological Science Institute Co. Ltd §Gene Research Center, University of the Ryukyus The study of gene technology has made remarkable in recent years; various conditions of DNA have been treated. As the study has developed, large size and quantities of DNA had been treated in the field of gene therapy and genome analysis. Especially, it has been remarkable to study large size and quantities of plasmid DNA, which is mainly used as material for the research in these regions. In the field of medicine, such as gene therapy, safer material of the research and purer plasmid DNA are required. It is difficult to deal with the DNA size which is becoming larger, and quality control which is getting severe, in the traditional way of the plasmid DNA. Therefore, it is necessary to figure out the way to get the larger and purer plasmid DNA which meets the needs of those two things. In the case of plasmid DNA extracted from a large scale of cultivated bacteria, there is the problem that the operation for the extraction is complicated because it should be solved only by dividing it into the small scale of extractions. In consideration of the need for gene technology, in the future, it will surely bring great benefit in this field to develop a simple and easy way of purer and larger size and quantities of plasmid DNA. In the purpose of developing the way to obtain such the plasmid DNA, we considered a new system, which is extracting plasmid DNA from bacteria with membranes not with centrifugation and in which line processes are adopted and column chromatographs are improved.

166. LONG-TERM EXPRESSION IN MOUSE LUNG AFTER NON-VIRAL SLEEPING BEAUTY-MEDIATED GENE TRANSFER Lalitha R. Belur, Joel L. Frandsen, Perry B. Hackett, R. Scott McIvor Gene Therapy Program, Institute of Human Genetics, Dept. of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 A number of different non-viral vector systems have been used for successful delivery of genes to the lung, but have been limited by the lack of long term gene expression. Transposons are mobile genetic elements and are useful gene therapy vectors, since in the presence of a transposase they bring about the integration of transgene sequences into the host chromosome. We have used the Sleeping Beauty transposon system to mediate long term gene expression in the lung. A transposon containing the luciferase gene under transcriptional regulation of the Rous sarcoma virus promoter (pTL) was complexed with linear PEI (ExGen 500, MBI Fermentas) with or without a second plasmid encoding the Sleeping Beauty (SB) transposase (pCMVSB10) and injected intravenously into C57BL/6 mice. 5 animals from each group were sacrificed at 24 hours, 2 weeks and 8 weeks, and the lungs were removed and assayed for expression of the luciferase reporter gene. Animals that received the luciferase transposon in the absence of pCMVSB10 exhibited no luciferase expression 2 months post-injection, while animals that received roughly equimolar amounts of the luciferase transposon and SB transposase plasmids continued to express luciferase activity at a level that was 100-fold above background and 5% that of the 24 hour luciferase expression level. In a separate experiment, C57BL/6 mice were injected via the tail vein with beta-galactosidase transposon (pTRSV␤Gal)-PEI complexes in the presence or absence of SB transposase plasmid in order to evaluate long term localization of transgene in the lung. Experiments to elucidate flanking sequences and assay molecularly for transposition are currently in progress. We have thus established conditions for sustained

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expression of newly-introduced gene sequences in the lung after intravenous delivery, dependant on co-delivery of SB10 transposase to mediate transposition. This gene delivery approach and the observed expression in the lung are potentially applicable to the treatment of a variety of pulmonary diseases.

167. Gene transfer into skeletal muscle by ultrasonography contrast agent microbubbles Katsuro Tachibana*, Hironori Abe†, Motomu Kuroki†, Masahide Kuroki† *First Department of Anatomy, Fukuoka University School of Medicine †First Department of Biochemistry, Fukuoka University School of Medicine, Japan Arterial occlusive diseases cause serious ischemic conditions in various organs, such as the heart, brain and leg. Therapeutic angiogenesis is believed to be beneficial for such conditions. Intramuscular injection of naked plasmid DNA encoding angiogenic growth factors offers a promising new approach for such purposes, however, only a small amount will pass through the cell membrane leading to low gene-transfer efficiency. Recent studies have shown that therapeutic ultrasound (US) alone can induce or increase cell membrane permeabilization of various agents including genes. It is currently suggested that the mechanism of this phenomenon is closely related to acoustic cavitation. This study addressed the hypothesis that commercially available ultrasonography contrast agent microbubbles could be used to increase gene transfection efficiency in vivo by relatively low intensity ultrasound-mediated microbubble destruction. Two different types of ultrasonography contrast agent microbubbles (0.02mL; Albunex or Optison, both Molecular Biosystems) were separately mixed together with a commercial reporter plasmid DNA (25ug; pGeneGrip, GTS Inc.) encoding green fluorescent protein (GFP) immediately prior intramuscular injection into mouse thigh quadriceps muscle. After the mouse was anesthetized, therapeutic ultrasound (1 MHz) was irradiated to the plasmid DNA injected muscle site at an intensity of 2.0 W/cm2 for 2 minutes. Mice were sacrificed 7 days after ultrasound treatment for gene expression assay. GFP distribution patterns were observed by confocal laser microscopy (Carl Zeiss) and quantified by computer image analysis. Muscle tissue irradiated with ultrasound in the presence of albumin-coated, air-filled Albunex microbubble revealed no difference of the number of GFP expressing muscle fibers as compared with ultrasound alone. In contrast, albumin-coated, octafluoropropane gas-filled Optison microbubble showed a 10fold increase in the number of GFP expressing fibers (ANOVA, p⬍0.05). No significant enhancement were observed with Albunex alone or Optison alone. Low intensity ultrasound increased the transfection of naked plasmid DNA within skeletal muscle in the presence of octafluoropropane-filled Optison but not in air-filled Albunex microbubbles. As Optison has a longer life span than Albunex as a bubble, gene transfection may be attributed to repeated or slower bubble destruction during ultrasound irradiation resulting in greater number of cell membrane poration. Although further investigation should be performed to find the optimal type of gas-filled microbubble, already commercially available ultrasonography contrast agent, Optison might be used as a modality for efficient gene delivery into cells for induction of angiogensis and treatment of various diseases in the near future.

168. In Vivo Transfection of Murine Intestine Using Naked Plasmid DNA Elizabeth Parks*, Blake J. Roessler* *Department of Internal Medicine, University of Michigan, Ann Arbor MI Numerous studies have indicated that “naked” plasmid DNA can be used to effect spontaneous transfection of specific cell MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

PHYSICAL METHOD BASED GENE THERAPY types in vivo. We hypothesized that naked plasmid DNA would also effect transfection of cells within the gastrointestinal tract following oral administration. The goal of this study was to demonstrate transfection of gastrointestinal tissues in vivo using viral(v) IL-10 (BCRF1 gene product that is a homologue of murine and human IL-10) as a functional reporter transgene cloned into a conventional CMV promoter based expression plasmid. Mice with a homozygous deficiency for IL-10 have normal lymphocyte development and antibody response, but can become anemic at an early age and develop chronic enterocolitis, which can affect both the small and large bowel, resembling inflammatory bowel disease (IBD), depending on environmental constraints. Mice housed in specific pathogen free (SPF) conditions, such as those in this study, often develop either limited or no IBD-like symptoms. Eight week old male, non-diseased IL-10 deficient mice (B10.129P2(B6)-IL-10(tmCgn)) were administered by gavage a dose 100ug or 300ug of highly purified pNGVL3-vIL-10 in sodium bicarbonate buffer daily for 4 consecutive days. Age and sex matched littermate controls were treated with an equal volume of buffer alone. All animals were maintained on a liquid rodent diet and housed SPF for the duration of the experiment and euthanized on day 5. Samples of whole blood, liver, spleen, proximal and distal small intestine were obtained and total protein lysates were made. Whole tissue lysates were analyzed using an ELISA specific for vIL-10 (limit of detection 31 pg/ml). IL-10 deficient mice treated with the pNGVL3vIL-10 demonstrated significant levels of vIL-10 expression in the small intestine, with distal small intestine values 2 fold greater as compared to proximal small intestine lysates. No vIL-10 expression above the levels of controls were observed in serum, liver, or spleen. No significant differences in levels of v-IL10 expression were noted between animals that received a dose of 100ug versus 300ug of pNGVL3-vIL-10. These results suggest that gastrointestinal cells can undergo spontaneous transfection following contact with expression plasmid DNA, and the preliminary dose response findings suggest a saturation effect consistent with receptor mediated endocytosis. Current studies are attempting to define the anatomic location of transfected cells using in situ hybridization techniques. Identification of transfected cells will allow us to perform experiments designed to further elucidate the mechanism of transfection in gastrointestinal cells. Future studies will examine effects associated with the addition of functional excipients (bile salts and cyclodextrins) to the sodium bicarbonate buffer.

169. Regulation of angiogenesis and matrix remodeling by localized, matrix-mediated antisense gene delivery Themis R. Kyriakides, Tristan Hartzel, Grace Huynh, Paul Bornstein Departments of Biochemistry and Medicine, University of Washington, Seattle, WA Implantation of biomaterials, such as glucose sensors, leads to the formation of a poorly vascularized collagenous capsule that can lead to implant failure. This process, known as the foreign body reaction (FBR), develops in response to almost all biomaterials and consists of overlapping phases similar to those in wound healing. Implantation of porous biomaterials, such as polyvinyl alcohol sponges, also leads to granuloma formation within the interstices of the sponge prior to encapsulation by the FBR. We asked whether delivery of an antisense cDNA for the potent angiogenesis inhibitor, thrombospondin (TSP) 2, would enhance blood vessel formation and alter collagen fibrillogenesis in the sponge granuloma and capsule. Collagen solutions were mixed with plasmid to generate gene-activated matrices (GAMs), and applied to biomaterials that were then implanted subcutaneously. Sustained expression of plasmid-encoded proteins was observed at two weeks and a month following implantation. In vivo MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

delivery of plasmids, encoding either sense or antisense TSP2 cDNA, altered blood vessel formation and collagen deposition in TSP2-null and wild-type mice, respectively. Non-treated implants, implanted next to GAM-treated implants, did not show exogenous gene expression and did not elicit altered responses, suggesting that gene delivery was limited to implant sites. This method of antisense DNA delivery has the potential to improve the performance and lifespan of implantable delivery devises and biosensors.

170. Gene delivery from polymeric injectable implants Rom E. Eliaz, Francis C. Szoka Jr. Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, California 94143-0446, U.S.A. A gene delivery system with the benefits of an implant but the ease of administration of an injection to the site of need was developed. This delivery system involves a simple preparation procedure and avoids an invasive technique such as surgery in its implantation or removal. We have developed an in-situ forming implant system that can be administered as a liquid using standard syringes and needles. Upon contact with body fluids, the liquid system coagulates to form a biodegradable solid implant for gene delivery. We present systems comprised of various genes and a water insoluble biodegradable polymer dissolved in a pharmaceutically acceptable water-miscible solvent(s). The physico-chemical properties of the polymeric implant system were studied and utilized to predict the release of genes from these systems. Changes in polymer composition, its weight percent in solution, molecular weight, solvent composition, and plasmid DNA loading level were optimized to provide formulations with the desired release rates and duration of release. Bioactivity of the released plasmid pCMVLuc was assessed using PEI transfection in cultural cells and demonstrated that the released plasmid retained 100% of the transfection activity of the non-encapsulated plasmid. The results demonstrate that these novel controlled release systems can be fabricated for the release of very high molecular weight plasmid DNA which could be an approach to plasmid-base gene transfer, tissue engineering, and may provide solutions to problems such as periodontal ligament detachment, alveolar bone resorption and furcation defects.

171. Systemic Protein Expression Following Sustained Gene Delivery from Microspheres and Novel Polymeric Networks Krishnendu Roy, Yu-Ching Wu, Patricia Chambers, Mary Lynne Hedley, Shikha Barman Zycos Inc., 44 Hartwell Avenue, Lexington, MA, USA There is growing interest in the development of non-viral gene delivery systems due to their versatile ability to resist nuclease degradation, modulate plasmid DNA release to affect gene expression, and their low clinical risks compared to viral systems. In situations where long-term gene expression is required to provide a therapeutic effect, pDNA released from these delivery systems must be optimized for concentration to achieve efficacious levels for the desired duration. Furthermore, the formulations developed must have ease of administration, flexibility of dosing and route-appropriateness. In this study, we have developed and compared two types of delivery systems for level and duration of systemic protein expression following intramuscular injection using a mouse model. They were: (a) biodegradable microparticles, wherein pDNA was co-encapsulated with PEG-DSPE (methoxy polyethylene glycoldistearoyl ethanolamine) in polylactide-co-glycolide (PLG; 50:50 L:G, ⬃12K Da) using a modified double emulsion solvent-evaporation technique, and (b) a polymer-network system, wherein pDNA was mixed with PEG-DSPE and mutually reacting polyeth-

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PHYSICAL METHOD BASED GENE THERAPY ylene oxide-derived polymeric components to form an injectable in-situ crosslinking formulation. Plasmid DNA encoding for human SEAP (secreted embryonic alkaline phosphatase) was used in all formulations to study levels and kinetics of serum protein expression. We have previously reported that co-encapsulation of an anionic lipid with pDNA in PLG microspheres, when administered intramuscularly, can enhance the immune response against the encoded antigen. However, for particle-mediated pDNA delivery, effect of different dosage and repeat injections on protein level and duration needs to be established. In this study, the roles of anionic lipid, pDNA dosage and injection regimens on gene expression via microsphere-mediated delivery were determined. In addition, an injectable, biodegradable polymer network formulated with pDNA was developed and gene expression levels and kinetics were established following intramuscular administration and compared to those obtained using microspheres. Microspheres were characterized for DNA and lipid content, DNA supercoiling and in-vitro plasmid release utilizing methods published previously. The polymeric network formulation was comprised of an equimolar combination (2: 2 %w/v) of two interacting PEG-based components admixed with pDNA before injection. Serum was collected at pre-determined time points via retro-orbital bleeding and assayed for bioactive SEAP levels using a luminometric kit. Expression levels of SEAP obtained via microparticles (⬍10um in size, 3.5-6 ug/mg DNA) were ⬃0.5-1 ng/ml (50 ug DNA dose) and ⬃1-1.5 ng/ml (100 ug DNA dose) sustained over 60 days. A repeat dose of microspheres at 60 days caused an increase in expression levels. Comparatively, SEAP levels generated utilizing the network formulation were higher, approximately 15-25 ngs/ ml, peaking at ⬃7-10 days and dropping after ⬃15 days postinjection. Optimization of both promising formulations to obtain enhanced and sustained gene delivery is underway.

172. Manufacturing Technology, Regulatory and Economic Impact by CMOs on the Advancement of Plasmid DNA-Based Drug Development John Vu*, Henry Hebel*, Astrid Breul†, Markus Muller†, Joachim Schorr† *QIAGEN Inc, USA †QIAGEN GmbH, Germany Ongoing research for over a decade in the field of gene therapy and genetic vaccination has driven an increasing number of gene-based drug candidates from research and pre-clinical stages to clinical investigation. As gene therapy projects progress to human clinical trials, the investigation takes on a substantially new dimension where manufacturing technology, GMP regulatory issues and economic feasibility can significantly impact the commercial realization of a drug candidate to the same extend as the evaluation of the drug safety and efficacy. To overcome such manufacturing technology, regulatory and economic challenges, many biotech and pharmaceutical companies have relied on contract manufacturing organizations (CMOs) to provide time and cost saving solutions to help expedite the development of gene-based drug in clinical research. CMOs such as QIAGEN Contract Manufacturing Services and its strategic manufacturing venture, pAlliance, are at the forefront of manufacturing technology development to meet safety concerns and regulatory changes while having the capability to provide large scale supply needs for clinical trial at a feasible cost to researchers. GMP manufacturing of gene-based drug for human consumption in clinical and commercial applications requires a process that produces highly homogenous product that is relatively free of undesired contaminations such as immunogenic and toxic substances. The process has to be able to manufacture product reproducibly from batch to batch and meet all the regulatory compliances for documentation and validation. The manufacturing process also takes into consideration the issue of scalability to accommodate the supply needs for all stages

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of clinical trial. The process has to be robust and scalable in order to ensure the reproducibility of the product in various batch scales within standard released specifications. In this presentation, as our expertise is mainly in production of non-viral vectors, we describe our latest manufacturing technology development work and the advancement of our scalable manufacturing process for plasmid DNA based- drug, regulatory and safety concerns from manufacturer’s point of view and the economic impact of current processing technology. We also present updates on current plasmid DNA manufacturing issues with the intention of helping researchers to aware of important points to be considered when dealing with GMP products and GMP regulations.

173. Using Real-Time Quantitative TaqMan PCR to Study Persistence of Plasmid Vectors in Mouse Lungs Ian Pringle, Lee Davies, Deborah Gill, Stephen Hyde GeneMedicine Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom Clinical studies for cystic fibrosis gene therapy have shown modest, short-lived, gene expression from plasmid DNA (pDNA) vectors in the airways. Similar results have been observed from pDNA delivered to the lungs of mice. A number of reasons for this short-lived gene expression have been proposed including, the rapid loss of transfected epithelial cells, the loss of vector DNA from cells, an immune response against the transgene product, or transcriptional silencing of the plasmid. We have constructed the plasmid vector, pCIKLux which contains the firefly luciferase gene as a reporter under the control of the human immediate early CMV promoter and the poly-adenylation signal from human SV40. Following intranasal instillation into mouse lungs, reporter gene expression was maximal 2 days after dosing (9451 ⫹/-2720 mRLU/mg protein), but was undetectable at day 7. Similar results have been obtained in SCID mice and with a vector expressing an endogenous murine gene, suggesting that the rapid loss of expression was not due to an immunological response to the transgene. Previously, improved duration was demonstrated with a derivative of pCIKLux, pCIKLux.IO in which the luciferase transgene and adenovirus type 2 E4ORF3 polypeptide are co-expressed. Luciferase expression can be observed for up to 12 weeks from pCIKLux.IO (⬃10% day 2 pCIKLux levels at day 84). Suggesting that cells expressing the transgene can persist in the airways for at least three months. Using a TaqMan quantitative DNA assay we studied how plasmid based gene expression related to the persistence of pDNA in-vivo, and we compared two different methods of gene transfer, naked DNA and plasmid/liposome complexes. BALB/c mice were dosed intranasally with naked pDNA (100␮g pDNA in 150␮l water) and with pDNA complexed with Genzyme Lipid 67 (66␮g pDNA/ 0.5mM GL67 in 100␮l). Mouse lungs were harvested at day 2, 7, 14, 28 & 56 (n⫽5) after dosing and total DNA was prepared using the Qiagen DNeasy kit. A vector specific TaqMan PCR assay was performed to quantitate levels of pDNA in each sample. The results were normalised for genomic DNA input relative to genomic ribosomal DNA. Results showed that after naked pDNA delivery there was a mean level of 0.7–1.6ng pDNA per lung between day 2–14. After delivery of the GL67/pDNA complexes 1000ng was detected at day 2, but there was a rapid reduction to 2.5ng at day 28. However, there were no significant differences in detection of the two plasmids at any later timepoint. In conclusion, pDNA can be quantitated after delivery to mouse lungs either as naked DNA alone or as DNA/liposome complexes. In the case of GL67, plasmid DNA levels reduced by approximately 20 fold at every timepoint measured after day 2. There was no difference in the persistence of the two plasmids, pCIKLux and pCIKLux.IO, even though gene expression from pCIKLux persists only for a number of days in-vivo while expression from pCIKLux.IO persists for 3 months. This supports the hypothesis that transcriptional silencing of plasmid vectors is a major factor in the short-lived gene expression observed in clinical studies. MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

PHYSICAL METHOD BASED GENE THERAPY 174. Effect of Partial Methylation on Gene Expression from Plasmid Vectors Ian Pringle, Andrew Rose, Deborah Gill, Stephen Hyde GeneMedicine Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom The CpG response is a serious inhibitory factor in the development of plasmid based gene therapy. Plasmid vectors carry sequences of prokaryotic origin, which are essential for their production in Eschericia coli, such as, antibiotic resistance genes and the ColE1 origin of replication. Prokaryotic DNA contains on average 4 times as many palindromic CG motifs (CpG) than mammalian DNA. This increased frequency of CpGs is recognised by pattern recognition receptors in mammalian cells and activates NK cells and B-cells leading to a broad range inflammatory response with the production of pro-inflammatory cytokines. The effects of the CpG response can be overcome by re-engineering plasmid vectors to reduce the number of CpGs or by the in-vitro methylation of plasmids with the bacterial CpG methylase M.SssI prior to in-vivo delivery. However, studies in cell culture and in-vivo have shown that methylation greatly represses levels of gene expression. We studied the effects of partial methylation of a series of plasmid vectors on gene expression in cell culture. Plasmids expressing the Firefly Luciferase reporter gene have been developed in which expression is controlled by a number of different viral or human promoters. The plasmids pCIKLux (CMV IE promoter), pCIKLux.IO (CMV IE promoter and Ad2 EF ORF3 element) and pUBLux (human UbC promoter) were methylated in-vitro using the methylases M.HpaII, M.HhaI, M.Dual (M.HpaII ⫹ M.HhaI) and M.SssI leading to methylation of 7.5 %, 7.5 %, 15 % and 100 % of CpG sites respectively. Plasmids were transfected into HEK293T cells and Lux activity measured after 2 days. As expected the experiments showed that the methylation of each of the Lux expressing plasmids by M.SssI had a serious inhibitory effect on gene expression in cell culture resulting in a 300 - 1000 fold reduction in Lux reporter activity. Methylation of pUBLux with M.HhaI and M.Dual reduced expression by 3 - 4 fold but methylation with M.HpaII had no effect on expression from this plasmid. The vectors pCIKLux and pCIKLux.IO have no sites for M.HhaI or M.HpaII in the CMV promoter but still show a 2 - 3 fold reduction in expression when methylated with M.HhaI and M.Dual. Methylation with M.HpaII has little effect on expression from pCIKLux and pCIKLux.IO. Interestingly, these results indicate that when plasmids are methylated at low levels, the repressive effects of methylation may be more dependent on the position of methylated sites within the vector than on the density of methylation. Published cell culture studies have shown that low levels of plasmid methylation can increase nuclear retention of plasmid DNA, although it may also have an inhibitory effect on gene expression. These studies have implications for the design of plasmid vectors to ensure optimum levels of gene expression with a reduced host inflammatory response.

175. Electrotranfection of human peripheral blood stem cells Lin-Hong Li, Philip McCarthy, Sek-Wen Hui Roswell Park Cancer Institute, Buffalo, NY 14263 A major obstacle of gene transfer into hematopoietic stem cells, a key step for many gene therapy and tissue replacement applications, is its low efficiency. Electroporation is effective in transferring large genes directly into the cytoplasm. It has been applied to transfect a variety of cells including non-phagocytic and non-proliferating cells such as peripheral leukocytes and stem cells, which are usually refractory to chemical transfection vectors. High cell mortality is responsible for the low efficiency of electrotransfection. Using human primary CD-34⫹ cells from peripheral blood as a model, we found that transfection-induced MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

apoptosis and post-pulse colloidal-osmotic swelling are two main factors for the poor transfection of these cells. Reducing postpulse colloidal-osmotic swelling by pelleting is effective in improving electrotransfection of many hematopoietic cell lines, such as NK-L cells, NFS-70 pro-B cells and L1210 subclone 3-3 cells. The transfection efficiency is up to 3 times greater when pulsed human primary CD-34⫹ cells are incubated in a pellet than that when cells are incubated in suspension, when apoptosis is suppressed. Electrotransfection-induced apoptosis of human primary CD-34⫹ cells is not a result of the electroporation process, since electroporation in the absence of DNA causes no additional apoptosis as compared to unpulsed cells. We believe that the effect is related to the large amount of exogenous DNA delivered by electroporation, and that these cells are particularly sensitive to DNA-uptake-induced apoptosis. By applying caspase inhibitors (B-D-Fluomethyl Ketone and Z-VAD-FMK), apoptosis is reduced by ⬃22% of cells. Human primary CD34⫹ cells also undergo apoptosis from depletion of cytokines in culture. Since cytokines are found to reduce apoptosis of unpulsed cells (by ⬃43%), but not of pulsed cells, and about 16% survive without cytokines, whereas caspase inhibitors improve the viability of both pulsed and unpulsed cytokinedepleted cells, we believe that there are two independent mechanisms of apoptosis. By using the post-pulse pelleting method to suppress colloidalosmotic swelling, and caspase inhibitors to reduced apoptosis, we achieved a ⬃25% transfection efficiency of human primary CD34⫹ cells. This effort brings the ex vivo electrotransfection efficiency within the reach of therapeutic applications.

176. In vivo and in vitro evaluation of small (< 250 bp) liver-specific promoters Naoki Hamajima, Nachimuthu Chinnasamy, Richard A. Morgan Clinical Gene Therapy Branch, NHGRI, NIH, Bethesda, MD We have designed and evaluated four types of small (⬍ 250 bp) promoters for liver-directed Hemophilia A gene therapy by single AAV vector system: 1) E1ML: hepatitis B virus enhancer I and adenovirus type 2 major late promoter; 2) E1TK: hepatitis B virus enhancer I and herpes virus thymidine kinase promoter; 3) AFP: variant-type of human alpha-fetoprotein promoter; and 4) MU1: mouse U1 small nuclear RNA promoter. The requirement for maximizing the insert capacity of AAV vector is very important in the case of potential Hemophilia A treatments, because of the small packaging capacity of this gene delivery system (⬍ 5.0 kb) and the size of the smallest factor VIII-expressing cDNA (4.4 kb). Although dual AAV vector system using rAAV heterodimeration has been developed to overcome this limitation, there still remain some disadvantages in this system such as the difficulty in simultaneously transducing a hepatocyte with two rAAV and the necessity in producing two rAAV. The four promoters were inserted into a ß-galactosidase-expressing plasmid, and were analyzed both in vitro and in vivo. E1ML showed the strongest promoter activity in human primary hepatocytes, Gunn rat hepatocytes, and two hepatoma cell lines (Hep-G2 and HuH-7), although E1TK was the strongest in non-liver cell lines (COS-7 and 293T). To evaluate the promoter activities in liver in vivo, we carried out the hydrodynamics-based transfection by systemic administration of plasmid DNA via tail vein in mouse. E1ML showed stronger promoter activity than E1TK in liver at all time points (1, 2, 4 and 7 days after injection) evaluated. Thus we demonstrate that E1ML is the strongest and most liver-specific of the four small (⬍ 250 bp) promoters that we have evaluated here. These data also demonstrate that the hydrodynamics-based transfection by systemic administration of plasmid DNA via tail vein in mouse can be a very efficient means for the evaluation of promoter activity in liver in vivo.

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PHYSICAL METHOD BASED GENE THERAPY 177. Regulated Interferon Alpha Expression Following IM Injection of Plasmid with Electroporation Jason G. Fewell, Valarie Florack, Ingrid Anscombe, Jerry Perrard, Vidya Mehta, Ronald V. Abruzzese, Martha French, Jeffrey L. Nordstrom Valentis, Inc. Recombinant inteferon alpha is the primary treatment for chronic hepatitis C virus infection. The current best treatment regimen (interferon with ribavirin) has a relatively low response rate that is attributed in part to the short half-life of interferon alpha in the circulation. Emerging therapies are interferons with covalently attached polyethylene glycol moieties (peginterferon) that are shown to have a longer half-life, sustained absorption and a slower rate of clearance. Clinical trials have indicated that use of peginterferon given once weekly is more effective than using non-modified interferon three times weekly. The ultimate goal of interferon therapy is to have long-term sustained expression with a minimum number of treatments. A potential method of achieving this goal is gene therapy. We have injected plasmid encoding human interferon alpha (hINF-␣) into the tibialis of mice followed by administration of electrical pulses (electroporation) to facilitate plasmid uptake. Using a plasmid driven by the CMV enhancer/promoter and injected plasmid doses of 1.0 ␮g to 100 ␮g we have achieved a dose response of hINF-␣ expression with peak levels of 16,458 ⫾ 897 pg/ml. Expression with electroporation was 70 fold greater than without electroporation (238 ⫾ 100 pg/ml). The ability to tightly regulate expression of an interferon gene therapy may be a key component of the safety profile. Accordingly, we have expressed hINF-␣ in mice using the GeneSwitchTM regulatory system. The GeneSwitch system is based on a truncation of the ligand-binding domain (LBD) of the human progesterone receptor that lacks the ability to respond to progestins but retains the ability to respond to synthetic antiprogestins not as antagonists but agonists. A ligand-dependent sitespecific transcription factor is generated by linking the modified LBD to a heterologous DNA-binding domain (GAL4) and a transcriptional activation domain (NF-␬B p65). The chimeric protein (the GeneSwitch regulator protein) is activated by hormonal concentrations of antiprogestin to form a homodimer. This in turn binds to the promoter of a target gene with 17-bp GAL4 consensus sequences, and activates its transcription. A two plasmid mixture was injected into the tibialis of mice. One plasmid encoded the GeneSwitch protein under control of the skeletal actin promoter and the other plasmid encoded for hINF-␣ containing the GAL4 binding sites linked to a consensus TATA box. Using this system hINF-␣ levels peaked ⬃24 hours after administration of mifepristone, the low molecular weight inducer. In mice, a pulsatile pattern of hINF-␣ expression was achieved over the course of 1 month with peak levels reaching 210 ⫾ 64.53 pg/ml. Future studies will focus on our ability to produce longterm regulated hINF-␣ expression at biologically relevant levels in mice and the ability to scale up this procedure into large animal models.

178. A Novel Electrode Design for Efficient Electroporative Gene Delivery Feng Liu, Leaf Huang Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh Recently, gene delivery in vivo using electroporation has became an area of greater research interest. The prospect of utilizing electroporation for gene transfer in vivo is evident based on the success of delivering genes in vitro and effective delivery of drugs in electrochemotherapy studies. Although the detailed molecular mechanism of electroporation is still not completely understood, there is agreement in the literature that the field strength induces some sort of metastable structure defect in the membrane which serves as a pathway from the extracellular space to the cell interior. Electroporation is a threshold phenomenon. The field

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strength necessary for gene delivery must exceed a threshold value. Moderate increases in the applied field strength resulting delivery; however, large increases of the applied field from this threshold result in tissue damage. Here, we report a novel design of an injectable electrode, which utilizes comparatively much lower electric field strength to achieve high transfection efficiency. The hypothesis of this design was based on two electroporation parameters we defined. One is the Effective Threshold Value (ETV), which is required for the electrotransfer and is defined by the cell and its environment. Another one is the Apparent Threshold Value (ATV), which is defined by the electroporater. ATV is usually much greater than ETV. Theoretically, the field strength is reversely proportional to the distance from its source. Therefore, when the field strength is delivered from the conventional electrodes to the injection site of DNA, the field strength is dissipated due to the distance between the injection site and the electrode. To compensate for the loss in the field strength, higher ATV has to be applied which results in toxicity to the tissue. A logical approach to decrease ATV is to directly deliver the field strength to the injection site. In this case, ATV is approximately equal to ETV such that gene transfer can be achieved at lower field strength. This hypothesis was supported by our data. The maximal level of gene transfer was achieved at as low as 50 V/cm of field using an injectable electrode, whereas 200 V/cm was required by using a conventional electrode to achieve the similar level of gene transfer. Histochmical analysis showed that no muscle damage was observed using the new electrode. However, severe damage was induced by using the conventional electrode. We conclude that the new injectable electrode represents a significant improvement in elctroporative gene transfer to tissues. Supported by NIH grants CA74918, DK54225, AR45925, DK44935 and by Muscular Dystrophy Association of America.

179. Sleeping Beauty-Mediated Transposition in Cultured Human Lung and Liver Cells and Murine Hematopoietic Cells Andrea Converse, Lalitha Belur, Joel L. Frandsen, Perry B. Hackett, R. Scott McIvor Gene Therapy Program, Institute of Human Genetics, Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN The Sleeping Beauty transposon system has shown evidence of the ability to introduce foreign genetic elements into many different vertebrate cell types in vitro, and into mouse liver and lung in vivo. We have demonstrated up to a six-fold increase in stable gene transfer frequency when a transposase-encoding plasmid (pCMVSB10) is co-transfected along with a transposon engineered to contain either a neomycin phosphotransferase gene or a mutant dhfr gene (conferring resistance to methotrexate) in human HeLa (cervical carcinoma) cells, murine NIH3T3 fibroblasts, human A549 (lung carcinoma) cells and human Huh7 hepatoma cells. In Southern analysis of individual, G418-resistant NIH3T3 clones, one or more transposition events was observable by the absence of plasmid sequences immediately flanking the transposon termini compared with a pattern of random plasmid integration in colonies transfected without transposase plasmid. To evaluate transposition in hematopoietic cells, we have established ratios of DNA complexed with polyethyleneimine (PEI) which optimize gene transfer and expression levels in the murine myeloid leukemic cell line 32DP210. Optimal parameter settings were also established for electroporation in 32DP210 cells. We are currently applying these conditions (PEI-mediated gene transfer and electroporation) to evaluate ex vivo introduction and transposition of the luciferase gene into mouse bone marrow cells subsequently transplanted into recipient animals. Results from these experiments will provide determination of the potential effectiveness of the Sleeping Beauty transposase system to mediate non-viral transfer of therapeutic genes into hematopoietic cell targets. MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

PHYSICAL METHOD BASED GENE THERAPY 180. Plasmid DNA Transfection in Rat and Mouse Muscle: Effect of pDNA Dose, Injection Volume and Electroporation J. Hartikka*, V. Bozoukova*, M. Sawdey*, A. Vilalta*, M. de las Alas†, D. Hannaman†, B. Ellefsen†, R. Bernard†, M. Manthorpe* *Vical Incorporated †Ichor Medial Systems, San Diego, California During the past several years, it has been shown that the ability of mammalian cells to express proteins encoded by injected naked plasmid DNA (pDNA) in vivo can be utilized to generate humoral and cellular immune responses against foreign antigens. However, there has been only limited success in using plasmid DNA-based delivery of therapeutic proteins in large animals and humans. It has been suggested that transfection with pDNA is inefficient and/or expression is low in large muscles. To enhance the therapeutic utility of pDNA, we have evaluated the effects of pDNA dose, injection volume and electroporation on pDNA transfection in rat quadriceps muscle, which is 8 – 10 times larger than mouse quadriceps muscle. First, we tested whether muscle transfection with pDNA is as efficient in rats and mice by comparing expression levels three days after intramuscular injections of pDNA encoding luciferase reporter. When either 10 ␮g of pDNA per 10 ␮l of vehicle or 50 ␮g of pDNA per 50 ␮l of vehicle was injected, expression in rat muscle was 5- to 10-fold lower than in mouse muscle. Injecting five times more pDNA in the same volume resulted only in a marginal 30% increase in expression in rat muscle. When 50 ␮g of plasmid was injected in 10 ␮l volume, expression in rat muscle was 4 ng luciferase per muscle. When 50 ␮g pDNA was injected in 50 ␮l or 500 ␮l volume, luciferase expression increased 8- and 21-fold, respectively. In order to study the distribution of expression in the target tissue, rat quadriceps muscles were cut longitudinally into ten 2 mm sections, and luciferase activity was measured from each section. These studies revealed that large injection volumes increased expression around the injection site and resulted in transfection throughout the longitudinal axis of rat quadriceps muscle. Large injection volumes increase muscle transfection with plasmid DNA, presumably by generating hydrostatic pressure and by distributing the pDNA solution over a larger area. Furthermore, lack of pressure might explain why expression in rat muscle is lower than in mouse muscle when identical pDNA doses and injection volumes are used. However, correspondingly large injection volumes cannot be used for intramuscular injections in humans. Therefore, we performed further studies in rat muscle using a 10 ␮l injection volume. This volume in rats, if scaled up on a body mass basis, is a more comparable volume to those currently used for human intramuscular injections. Injecting 10 ␮g of pDNA in 10 ␮l vehicle resulted in an expression level of 3 ng luciferase per muscle. When identical injections were done in combination with electroporation, average expression in rat muscle increased by 170-fold to 560 ng. Thus, electroporation greatly enhances muscle transfection with pDNA when small injection volumes are used. Electroporation might be used to enhance plasmid DNA delivery in clinical gene therapy applications.

181. High ionic strength enhances dermal gene transfer Sophie Chesnoy, Leaf Huang Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh Recent development of electroporation as a gene delivery method for introducing genes into the epidermis and the dermis of skin is very promising for potential gene therapy applications for skin diseases. Electroporation consists of the transient opening of pores in cell membranes exposed to short duration high voltage electric pulses. As a result, gene transfer is significantly improved because large amounts of DNA can diffuse through MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

these pores and enter the cell. Here, we report that dermal gene transfer in mice can also be greatly enhanced, up to the same extent as with the use of electroporation, by simply intradermally injecting a high ionic strength solution of naked DNA. By this method, the location of gene expression was found mainly in the smooth muscle cells underlying the skin and in the epidermis. As control, intradermal injection of an isotonic DNA solution only resulted in the transfection of the smooth muscle cells. In comparison, adipocytes in the hypodermis and keratinocytes in the dermis were also transfected in addition to the smooth muscle cells and the epidermis when electroporation was used. The improved transfection efficiency observed by intradermal injection of high ionic strength DNA solution was associated with some local, but reversible inflammation. Finally, the expression of a murine IL-12 plasmid was assessed by both methods and the results showed comparable gene expression levels and biological activity of the plasmid. Supported by NIH grant AI4885, DE13059, CA74918.

182. In Vivo Gene Expression in Muscle Using Nonionic Polymer Delivery System Jijun Wang, Elizabeth Wilson, Isaac Van Sligtenhorst, Michelle Fix, Mike Coleman Valentis, Inc., The Woodlands Center, 8300 New Trails Drive, The Woodlands, TX 77381, USA Numerous studies have demonstrated that genes can be transferred into striated muscle cells following the intramuscular (i.m.) injection of purified plasmids in isotonic saline (“naked” DNA). However, the bioavailability of “naked” DNA is very low and the resulting gene expression has been found to be highly variable. We have previously demonstrated that formulation of plasmids with certain non-ionic polymers such as PVP and PVA can increase plasmid delivery to skeletal muscle and reduce variability. We have observed that certain properties of the PINC (Protective, Interactive, Non-Condensing) polymers may account for these results in muscle. In studies reported here we demonstrate that other non-ionic PINC polymers, poloxamers, can significantly enhance plasmid delivery to skeletal myofibers after direct intramuscular administration. Various poloxamers were investigated in this study to determine their ability to enhance plasmid delivery to skeletal muscle and for their effects on plasmid stability and lyophilization. To determine effects on gene delivery of an expression plasmid encoding for luciferase was formulated with the poloxamer and a 10-␮g dose was injected into the tibialis anterior muscles of male CD-1 mice. Injected muscles were harvested 7 days post-treatment and assayed for luciferase activity. Formulation with poloxamer enhanced gene expression within the muscle by 2-30-fold compared to naked DNA. Among the poloxamers tested poloxamer 124 elicited the largest increase in gene expression followed by poloxamer 188 ⬎ 237 ⬎ 338 ⬎ 407. Histological analysis of tibialis muscles that had been injected with plasmid encoding Green Fluorescent Protein (GFP) formulated in poloxamer 188 confirmed transgene expression in a greater fraction of myofibers relative to naked DNA. Various formulation parameters that could affect plasmid delivery such as polymer concentration, molecular weight (for different poloxamers with similar of EO/ PEO ratio), HLB value, and salt/buffer concentrations, were investigated in our studies. Area under Curve (AUC) analysis of transgene expression indicated the formulation of plasmid with poloxamer 188 increased expression at least 5-fold over that of naked DNA for 21 days. No abnormal pathological changes were observed in muscles injected with plasmid formulated with the poloxamer. A lyophilized formulation of plasmid with poloxamer 188 remained stable for at least one year at 4°C. Based on results of these studies, formulation with poloxamers, such as poloxamer 188, appears to yield a stable plasmid formulation for enhanced delivery to skeletal muscle.

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PHYSICAL METHOD BASED GENE THERAPY 183. Modulation of Gene Expression Intramuscularly by Polymer Network Formulations Krishnendu Roy, Yu-Ching Wu, Patricia Chambers, Mary Lynne Hedley, Shikha Barman ZYCOS INC. 44 Hartwell Avenue, Lexington, MA 02421 Expression of genes at therapeutically efficacious levels presents an attractive mode of treatment of many disease etiologies. However, a major technical impediment has been achievement of effective gene transfer into cells, to generate high expression levels of the therapeutic gene. Although methods such as liposomal delivery, complexation with cationic lipids, etc. have been shown to work with mammalian cell cultures in-vitro, challenges exist in effective introduction of genes to target cells in-vivo. High transfection is achieved with DNA delivery with viral vectors, but these delivery systems have safety issues such as host response to the virus, oncogenic and inflammatory effects as well as dependence of gene introduction on the viral infection pathway. These issues have led to the development of synthetic gene carriers such as microparticles and DNA-polymer complexes. Dosing via microparticles ⬍10 um while targeted to cells by phagocytosis, is limited by DNA encapsulation efficiency and maximum allowable particle intake. We introduce synthetic polymeric networks structurally designed to modulate pDNA release and therefore, gene expression by control of formulation composition to affect network density. The formulations were optimized to be injectable, forming networks post-injection into muscle tissue. The formulations were comprised of polyethylene oxide-derived mutually reacting polymers containing pDNA (100 ug) and an anionic lipid (polyethylene glycol-distearoyl ethanolamine), mixed before injection intramuscularly into mice (100 ug DNA dose; tibialis muscle). A plasmid encoding for human SEAP (secreted embryonic alkaline phosphatase) was utilized to measure gene expression in serum. The formulations were comprised of equimolar concentrations of each component (2: 2, 5:5, 8:8 % w/v), with DNA and lipid added to one of the components. Network densities were characterized by in-vitro swelling in PBS at 37°C. Compatibility of DNA with the components was determined by incubation of all components at 37°C for 1 hour; percent DNA supercoiling post-incubation was measured by gel electrophoresis. Network formation in muscle was confirmed by injection of the formulations, then dissection of the tissue. Formulations 5:5 and 8:8 %w/v were crosslinked hydrogels, while 2:2 % w/v formulations were viscous liquid. Plasmid DNA retained supercoiling when incubated with the polymers, demonstrating chemical compatibility of all formulation components. SEAP levels were measured in serum utilizing luminometry at pre-determined timepoints and plotted in ngs/ml versus time in days. SEAP levels were highest at 7-10 days (⬃15-25 ngs/ml) for the 2:2 formulations, the lipid-containing composition expressing slightly higher than the non-lipid compositions. 5:5 %w/v formulations with incorporated DNA generated SEAP levels that were lower (⬃1-1.5 ngs/ml), sustained long-term. 8:8 %w/w formulations were ⬃0.5 ngs/ml SEAP in serum, comparable with saline controls. Regulation of gene expression via control of network density of these formulations is further being optimized for level and duration.

184. A Novel Method For Site-Specific Dual Isotope (3H/14C) Labeling Of Plasmid DNA Gururaj A. Rao*, Joseph Rabinowitz†, Richard J. Samulski†, Dhiren R. Thakker* *School of Pharmacy, Division of Drug Delivery and Disposition, University of North Carolina at Chapel Hill †Gene Therapy Center, University of North Carolina at Chapel Hill A method for selectively introducing two different radionuclides (3H/14C) into specific regions of plasmid DNA was developed. This method is based upon the ability of bacterial methylases to

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Figure shows a representative image of 14C labeled plasmid constructs. The scale on the right shows the relative intensities of the bands. The numbers on the left denote length of the fragments.

transfer methyl groups on to specific restriction sequences of DNA. Plasmid DNA was modified to contain sites that were targets for methylation by either the EcoR I or BamH I methylase. A 55-mer oligonucleotide, containing 6 repeats of EcoR I target sites (GAATTC) and its complement were synthesized and annealed (EcoR I insert). Similarly a duplex containing 7 repeats of BamH I target sites (GGATCC) was also prepared (BamH I insert). The EcoR I and BamH I inserts were cloned into the pCAT plasmid vector, downstream and upstream of the Chloramphenicol Acetyl Transferase (CAT) gene, respectively. Methylation of the target sequences in the plasmid constructs was carried out by EcoR I and BamH I methylases in the presence of [14C] or [3H] S-Adenosyl Methionine (SAM) as the methyl group donor. After methylation, the plasmid DNA was subjected to restriction digestion and gel electrophoresis/ phosphorimaging analysis. It was observed that greater than 80% of the radioactivity was localized in the fragments containing the target sequences (⬃500 base pairs in length, see figure). In contrast, the same fragment did not show any radioactivity for the unmodified pCAT control vector. This confirmed that the method developed allows selective incorporation of radioactivity (3H/14C) into specific regions of plasmid DNA. Experiments were also conducted to determine possible causes for the incorporation of label into non-target regions of the plasmid. Preliminary experiments indicated that this was not due to entrapment of the radioactivity in the larger fragments. Scanning the sequence of the pCAT-3 vector showed several sites that were related to either the EcoR I or the BamH I target sites. The possibility that these related sites might be targets for non-specific transfer of methyl groups, thereby contributing to the radioactivity in the rest of the plasmid, will be investigated. This method of radioisotope labeling is likely to be useful for studying in vivo bio-distribution of plasmid DNA and its metabolic fragments.

185. Factors important for efficient transposition from Sleeping Beauty-based vectors in vitro and in vivo Leonard Meuse*, Stephen R. Yant*†, Shiliang Shen*, Mark A. Kay* *Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA †Program in Molecular and Cellular Biology, University of Washington, Seattle, WA The transposable element Sleeping Beauty (SB) is currently under development as a new integrating vector system for basic reMOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

PHYSICAL METHOD BASED GENE THERAPY search and human gene therapy applications. Previously, we reported that transposase overproduction in mouse liver resulted in decreased levels of transposon-based gene expression in the long-term (Yant et al., Nat. Genet. 25:35-41). Herein, we demonstrate that regardless of transposase concentration or its catalytic activity, there is no detectable liver toxicity in vivo that is associated with transposase expression in either normal or immunecompetent scid mice. Since a number of related transposases are known to be regulated by a process called overproduction inhibition, we have employed a PCR-based approach to test whether the relative frequency of transposon excision in mouse livers varied according to transposase gene dosage. Results indicate that Sleeping Beauty’s activity can be significantly impaired at high enzyme concentrations in vivo. Furthermore, these studies, in combination with the direct isolation of chromosomal-transposon junction fragments from the livers of DNA protein kinase (DNA-PK cs)-deficient scid mice, suggest that DNA-PK-dependent double-strand break repair pathways are not required for transposition in mouse liver. In separate studies, we have analyzed the cell cycle status of mouse hepatocytes undergoing transposition by BrdU-cytoplasmic lacZ co-localization in mice. Results demonstrate that Sleeping Beauty can function equally well in both dividing and nondividing (quiescent) mouse hepatocytes, suggesting that cell-cycling is not required for transposition in mouse liver. Finally, by comparing the transposition efficiency of linear elements with and without flanking transposon repeats in cultured cells stably expressing the SB transposase, we show that linearization of donor transposon DNA greatly impairs the frequency of transposition in vitro. Based on these experimental findings, we have developed a modified helper-dependent adenoviral vector encoding both transposase and transposon functions in order to facilitate the in vivo delivery of this integrating system. The general utility of this vector is currently being assessed in vitro and in vivo. This work was funded by NIH grant DK49022.

186. Electroporation Can Enhance Naked DNA Transfection in The Spleen Xi-Zhang Lin*, Chung-Min Jen*, Chiung-Yu Jen*, Chung-Jen Huang*, Hsiao-Sheng Liu*, Ning-Sun Yang† *National Cheng Kung University Hospital, Tainan, Taiwan †Institute of Bioagriculture, Academia Sinica, Taipei, Taiwan Injection to the spleen is an effective approach to the lymphocytes. It is a feasible and safe clinical procedure as perform in splenoportography. As naked DNA injection is effective to transfect the liver through the tail vein, if hydrodynamic pressure is well manipulated, we try to inject reporter genes into the spleen to see if naked DNA injection is effective for gene transfer to lymphocytes. In addition, electroporation is used to compare the efficiency of a simple injection with and without this procedure. Green fluorescent protein and luciferase gene with CMV promoter was prepared for naked DNA injection. Three groups of Wister rats (Control 3, simple injection 6, injection with electroporation 6) were used for experiments. After adequate anesthesia, we do laparotomy to expose the spleen. Then, 100 ␮g DNA with d. d. water was injected into the spleen from lower pole with 28 G needles rapidly. During the injection, we carefully try to avoid leakage. For electroporation group, two plates (about 0.5 cm wide, 4 cm long) were used as electrodes from our laboratory. The plates were compressed gently over the spleen from anterior and posterior sides. Four series of 100 volts, 50 ms electric shocks were delivered. The rats in the control group were injected with normal saline. After the procedure, the abdomens were closed. In the green fluorescent protein experiment, the rats were sacrificed two days later and the spleens were removed for frozen tissue examination under fluorescent microscopy and hematoxylin and eosin staining; in the luciferase experiment, the spleens were removed for the activity measurement. We found that both naked injection and injection with electroporation did good transfection expression under microscopic examinations, while the conMOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

trol groups did not. The expressions were more prominent among germinal centers. For luciferase activity measurement, control group was 3.9 ⫾ 2.6 RLU/mg proteins, simple injection 55.6 ⫾ 24.3 RLU/mg protein, and injection with electroporation 552 ⫾ 301 RLU/mg proteins. Comparing with control group, both experimental groups expressed the activity significantly; moreover, comparing with the simple injection group, electroporation can enhance the efficiency significantly (p ⬍ 0.05, by nptrend test from Stata software) by about 5 to 40 folds. Our approaches are effective for the gene transfection to the spleen. Because the plates used for electroporation can be modified for the laparoscopy, both simple injection and injection with electroporation can be applied into clinical practice and have great potential for gene therapy.

187. Developing Multiple Needle Microseeding Techniques for DNA Vaccination: A Pilot Study Using the Foot-and Mouth Disease Virus System Ku-Feng Lin, Jeng-Hwan Wang, Chien-Yu Wang, Ning-Sun Yang Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan DNA transfer into skin is an attractive approach for gene-based vaccines, due to several considerations, including: 1) large and exposed tissue size or areas for gene delivery, 2) the dermal and cutaneous microenvironments are rich in professional antigen presenting cells (APCs) and accessory cells capable of initiating and facilitating various immune responses, and 3) level of transient transgenic proteins can be non-invasively detected (see below), 1-2 days after immunization to potentially project and fine tune future vaccination effect. These considerations appear to make the skin tissue a unique and promising tissue for expression of transgenic antigens and induction of immune responses. In this study, we report a new technical approach for in vivo delivery of naked DNA into epidermal and dermal tissues of test animals including mice and pigs. The microseeding system is made from a simple device where a group of 20 to 50, solid, very thin, accupuncture needles were bundled together, and they were administered into target skin tissue in a steady and continuous motion. Before those multiple needle microseeding treatment, candidate vaccine gene constructs can be topically applied to skin as viscous and concentrated solution in small volume. With this method, the expression levels of transgentic luciferase gene were shown to be similar or comparable to the gene gunmediated, intra-dermal injection, and intra-muscular injection for DNA delivery. We have observed and report here that immunizing mice with 5 microgram of FMDV coat protein (VP4-1) cDNA expression vector, three times by microseeding (each with two-week as interval) can effectively induce the production of transgenic VP1 coat protein in the targeted skin tissue and the production of neutralizing antibodies (NA) against FMDV in serum of test animals. This is, to our knowledge, the first demonstration of the use of a polycistronic FMDV cDNA for vaccination without the inclusion of a viral protease or the encoding gene by a DNA vaccine strategy. In addition, we also evaluated the capacity of transgenic VP1 antigen expression by using a tape for non-invasive, fast and simple collection of transfected epidermal cells. Cells of topical epidermal layers can be effectively and conveniently peeled off from test mouse skin after 2 days of DNA immunization by using an industrial brand, transparent tape without having to surgically sacrifice any of the test mouse skin tissue. These preliminary results show that the current gene delivery method may be practical, effective and convenient for enhancing and monitoring transgenic expression levels in vivo, and may result in improved techniques and strategies for future application to in vivo gene delivery, gene-based vaccination and gene therapy.

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PHYSICAL METHOD BASED GENE THERAPY 188. The Effect of Dosing Interval on Reporter Gene Expression in Mouse Lung Deborah Gill, Ian Pringle, Lee Davies, Stephen Hyde GeneMedicine Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, UK We have previously observed that reporter gene expression following delivery of plasmid DNA (naked or complexed with DCChol/DOPE, or GL-67) to the airways of mice is transient, diminishing to background levels within ⬃1 week of dosing. One approach to extending reporter gene expression is to re-administer the gene transfer reagent, although published studies suggest that such re-administration is limited at least in part by an anti-transgene immune response. By varying the interval between doses we aimed to identify a ‘dosing interval’ that would allow sustained plasmid DNA-mediated reporter gene expression. Plasmids expressing different transgenes were used to avoid possible complication with anti-transgene immune responses, such that animals were initially dosed with pCIKCAT (CMV based CAT expression vector) and then again at either 14, 28 or 42 days with pCIKLux (CMV based Lux expression vector). Plasmid vectors (100␮g pDNA in 150␮l water) were administered intranasally to female BALB/c mice (6-8 weeks old at the time of first dose) following methoxyflurane (Metofane) anaesthesia. Two days after the final dose, lungs were harvested and processed for Lux activity. The results showed that a dosing interval of 28 days or greater allowed reporter gene expression on a second dose (10488⫾2928 mRLU/mg) to be equivalent to animals that have only received a single dose (9452⫾2721 mRLU/mg; Mann-Whitney U. P⫽0.949). However, allowing only 14 days between doses, markedly reduces the efficacy of a second dose (2936⫾1224 mRLU/mg; P⫽0.048). A further study was conducted to determine which component of our gene transfer reagent was limiting efficient re-administration at 14 days. Groups of animals were dosed with pCIKCAT, pCI (‘empty’ vector), vehicle only, anaesthesia only, or remained untreated. After 14 days, all animals were dosed with pCIKLux and lungs processed for Lux activity 2 days later. Compared to the untreated group, all other groups (including anaesthetic only) showed a reduced gene expression after the second dose (P⫽0.049). We conclude that the process of anaesthesia alone is responsible for the reduced gene expression following a second dose at 14 days. The reason for this is unclear, but it is known that the inhaled anaesthetic, methoxyflurane, is metabolised to toxic by-products which remain present in plasma for up to 10 days after administration. Further studies may clarify whether the reduced gene expression is due to less successful administration of the formulation, reduced dispersal within the airways, or a reduction in cellular uptake of plasmid DNA. We conclude that re-dosing experiments using methoxyflurane should avoid re-dosing within 28 days, or alternative anaesthetics used. Clearly, anaesthesia can have a profound impact on reporter gene expression in re-administration studies.

189. Hydrodynamic Non-Viral Transfer of the Human Growth Hormone Gene into SCID Mice leads to Increased Body Weight Henrik Holst*, Frederik Dagnaes-Hansen†, Uffe Jensen*, Thomas Jensen* *Institute of Human Genetics, University of Aarhus, Denmark †Department of Medical Microbiology and Immunology, University of Aarhus, Denmark Non-viral gene transfer into the liver have generally been associated with low levels of expression and short duration. In mice, however, studies showed that rapid injection of naked plasmid DNA in a large volume into the tail vein of mice leads to high levels of gene expression in the liver (Liu et al, Gene Ther 6,125866,1999; Zhang et al, Hum Gene Ther 10,1735-7, 1999). We have found that rapid injection into the tail vein of DNA in a large volume (up to 8 % v/w of the body weight) leads to high expression levels in the liver of the multimeric protein mannan-bind-

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ing lectin (MBL), an essential component of the innate immune defence system (Vorup-Jensen et al, submitted). In order to obtain prolonged expression after hydrodynamic gene transfer into the liver, we compared expression constructs based on various viral sequences (AAV, EBV) and promotors (viral and endogeneous) using the human growth hormone gene as the reporter gene. Using a strong endogeneous promotor (the ubiquitin promotor) and a human growth hormone gene containing introns, inserted into a simple plasmid vector, we have been able to demonstrate high levels of human growth hormone expression (more than 80 ng/ml) for more than 4 weeks. Mice expressing the highest levels of the protein increased markedly in body weight during the first four weeks.

190. The R4 phage integrase as an integration tool for gene therapy vectors Eric C. Olivares, Roger P. Hollis, Bhaskar Thyagarajan, Amy C. Groth, Daniel S. Ginsburg, Michele P. Calos Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120 One approach to obtaining long-term expression from gene therapy vectors is to integrate them into the genome of target cells. For optimal safety and effectiveness, integration should be targeted to specific sites that are well-expressed and do not disrupt genes. We are using phage integrases to bring about site-specific integration in mammalian cells. We have demonstrated that the integrase from Streptomyces phage ␾C31 works in mammalian cells and can bring out efficient genomic integration at endogenous sites that resemble its wild-type attP recognition site. We have now shown that a second phage integrase, that of the Streptomyces phage R4, also works well in mammalian cells and can be used to efficiently integrate gene therapy vectors into the genome. We discuss the nature of the endogenous sequences that mediate this reaction and demonstrate how the ␾C31 and R4 integrases can be used sequentially to generate high integration frequencies. The types of integration cassettes under development can be used in connection with most gene therapy vectors to produce efficient site-specific genomic integration.

191. Extrachromosomal stable vectors for gene therapy Stephanie M. Stoll, Christopher R. Sclimenti, Edward J. Baba, Andrew S. Neviaser, Leonard Meuse, Mark A. Kay, Michele P. Calos Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120 We are interested in the potential of plasmid DNA that remains extrachromosomal in target cells to provide long-term high-level expression of therapeutic genes. We have constructed EBV/human plasmids that carry components of Epstein-Barr virus (EBV) plus human genomic sequences. In these studies we combined the EBV EBNA1 gene and its family of repeats binding sites on plasmids with human factor IX (hFIX) or human ␣1 antitrypsin (hAAT) sequences. Plasmids carrying these therapeutic genes and EBV components were delivered to mouse liver via hydrodynamic tail vein injection. This simple procedure, developed by J. Wolff and D. Liu, delivers plasmid DNA to up to 40% of hepatocytes. Gene expression was monitored by serum ELISA over a six month time course, with the result that these gene therapy vectors provided long-term expression of the therapeutic genes at levels that fully restore normal concentrations of these secreted gene products to the blood (10 ␮g/ml for hFIX and 1 mg/ml for hAAT). We believe that the EBV components, together with genomic elements such as genomic promoters and intronic sequences, combine to produce stable gene expression that does not become transcriptionally silenced. This work demonstrates that a single injection of naked plasmid DNA can provide effective, long-term, complete gene therapy, provided that the plasmid vector carries sequences that ensure long-term gene expression. MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

PHYSICAL METHOD BASED GENE THERAPY 192. Improved production of minicircle DNA in recAEscherichia coli strain with tightly controlled singlecopy Cre recombinase gene expression Oleg Tolmachov, Iwona Palaszewski, Brian Bigger, Michalis Fragos, Charles Coutelle Imperial College School of Medicine, Gene Therapy Group, London, United Kingdom Non-viral gene transfer is often impeded by cellular immunotoxic response to bacterial DNA sequences such as unmethylated CpG islands within the delivery plasmid. One way to avoid the presence of a bacterial backbone is to remove the bacterial sequences from delivery vectors before transfection to create DNA minicircles, which only contain the eukaryotic expression cassette. Such minicircles are likely to be less immunogenic and are smaller than the comparable plasmid vector thereby permitting higher transfection levels. We developed two recA⫹ and one recA- E.coli strains harbouring an AraC controlled chromosomally located Cre recombinase gene. We used these strains to excise the bacterial backbone flanked by mutant loxP66 and loxP71 sites in our plasmid. The resultant minicircle has a 34 bp hybrid loxP66/71 site which is poorly recognised by Cre recombinase and therefore equilibrium in the Cre reaction is shifted towards minicircle production. We found that minicircle DNA is produced more efficiently in a recA- strain where homologous recombination between minicircle and producer plasmid is blocked. We also present our method for construction of recACre-expressing strain where we have avoided the use of transposable elements which can potentially compromise therapeutic quality of the minicircle DNA.

193. Non-viral Gene Transfer into Resting Primary Lymphocytes: Small and Large Volume Linhong Li, Sarah Wang, Maria Wood, Vin Singh, Joe Fratantoni, Linda N. Liu TheraMed, Inc., 9640 Medical Center Drive, Rockville, MD 20850 Genetic alteration of peripheral blood T lymphocytes has been shown to have potential therapeutic application for inherited and acquired immuno-disorders. Resting lymphocytes are refractory to Moloney murine leukemia virus (MuLV) based gene transfer, the most common gene delivery system, because of their quiescent status. Here we report results of non-viral gene delivery to non-stimulated, resting primary lymphocytes in small and large volume, performed by electroporation. Resting primary lymphocytes were obtained from healthy donors’ apheresis white blood cells by standard Ficoll-Paque gradient and removal of adherent cells. Purity of the isolated lymphocytes was more than 90% as determined by immunophenotyping and FACS. Cell cycle analysis showed that 99% of the cell population was at G0/G1. First, we examined electroporation of the primary lymphocytes with macromolecules, FITCdextran (500,000 MW). Seventy-five percent of the cell population was loaded with FITC as determined by FACS, with 80% viable cells. Next, we tested electroporation of standard mammalian cell expression vectors, pCMV-eGFP, pCMV-Ds-Red, pCMVLacZ. A total volume of 15 to 20 ul containing 20x106 cells/ml and 0 to 1000 micro-gram plasmid DNA/ml were tested in a static chamber. Transgene expression was dose dependent on the DNA concentration. Electroporation of transgene eGFP showed 50% of the cell population expressed the transgene, and about 70% of the cells were viable. pCMV-Ds-Red labeled about 30% of the cell population with 60% viability. pCMV-LacZ, whose size is bigger than the previous two transgenes, can transfect about 15% of the cell population. Similar results were obtained with transgenes expressing proteins with therapeutic applications. A flow, large volume electroporation-based closed system has been developed to meet the criteria of clinical, on-site, genedelivery. Currently, 2 ml of the cell volume at 50x106 cells/ml together with 400 micro-gram of pCMV-Ds-Red were tested. More than 12% of the cells were transfected with Ds-Red. In MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

conclusion, we can transfect more than 100x106 primary quiescent lymphocytes with transgenes, or electro-load them with macromolecules, in a single, closed process. This simple, safe and fast gene/macromolecule delivery system has the potential to be a useful tool for gene therapy and drug administration.

194. POLOXAMERS ENHANCE PLASMID DNA-BASED GENE EXPRESSION AND VACCINATION Loretta Sukhu, Mary Wloch, Carol Buchner, Jukka Hartikka, Rohit Mahajan, Diane Hazard, Mike Sawdey, Michal Margalith, Jackie Stupack and Marston Manthorpe Vical Incorporated, San Diego, CA Naked plasmid DNA is being evaluated in preclinical and clinical studies as a novel non-viral vector for delivery of protein vaccines or therapeutics. Plasmid DNA medicines may achieve better efficacy if the DNA can be formulated for high-level protein expression. In the decade since the first publication showing that plasmid DNA injected into skeletal muscle is expressed at low levels in myofiber cells [Wolff et al., Science 247: 1465 (1990)], we have increased intramuscular expression by over 2,600-fold by making improvements in the DNA vector [Hartikka et al., Hum Gene Ther 7: 1205 (1996)] and DNA vehicle [Hartikka et al., Gene Ther 7: 1171 (2000)]. Here, we evaluated whether plasmid DNA expression in muscle could be further enhanced by the addition of standard human drug excipients to the optimized vector-vehicle formulation. In a standard screening assay, 50 ug of VR1255 luciferase DNA was formulated in 50 ul of 150 mM sodium phosphate with or without an excipient and injected into BALB/c rectus femori followed 7 days later by extraction and assay of intramuscular luciferase activity. In this study, we evaluated 24 commercially available poloxamers (BASF) using a 0.001% - 10% dose range grid. Thirteen of the 24 poloxamers elicited a statistically significant 4 to 7 fold increase in luciferase expression. Optimal concentrations of poloxamer varied between 0.001% to 4%. Selected poloxamers at 0.01% concentration enhanced intramuscular expression of DNAs encoding non-secreted bacterial beta-galactosidase (beta-gal) and secreted forms of human placental alkaline phosphatase, murine erythropoietin and canine clotting factor IX and enhanced luciferase 5-fold in rat rectus femoris muscle. Poloxamers also increased serum anti-beta-gal antibody titers by 30-fold. Histological examination of mouse rectus femori revealed that poloxamer increased the number and intensity of staining of beta-gal-positive myofiber cells. Under the optimal conditions using 0.01% 25R2 poloxamer, more than an average 50% (n⫽18) of the rectus femoris myofiber cells expressed beta-gal DNA. Thus, the inclusion of low concentrations of poloxamer can enhance the expression of reporter and therapeutically relevant plasmid DNAs.

195. HER2 DNA immunization with costimulatory molecules elicits functional anti-HER2 antibodies Nathalie Scholler*, Mary L. Disis†, Amber Dahlin*, Janice Pullman*, Karl Erik Hellstrom*, Ingegerd Hellstrom* *Pacific Northwest Research Intitute, Seattle WA †University of Washington, Department of Oncology, Seattle WA Anti-tumor therapy using antibodies directed against HER2 mediate an effective anti-tumor response in combination with chemotherapy. Investigations in neu transgenic (neuTg) mice have also demonstrated that HER2 DNA immunization protects animals from the development of HER2 expressing tumors. Yet, data from human clinical trials with DNA vaccines have not convincingly demonstrated any efficacy in preventing or treating infectious disease or cancer. We hypothesized the addition of DNA encoding costimulatory molecules to a HER2 based DNA vaccine may improve vaccine efficacy. Using a pLNCX vector, we constructed DNA vaccines encoding full-length Rat Neu (pLNCX RN), murine CD80 (pLNCX B7.1), CD86 (pLNCX B7.2), and 4-1BB ligand (pLNCX 4-1BBL). We immunized neuTg mice with pLNCX RN alone or together with other plasmids encoding co-

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PHYSICAL METHOD BASED GENE THERAPY stimulatory molecules. No anti-HER2 antibodies were detected with pLNCX RN alone. In contrast, HER2 specific antibodies were observed in the sera of all mice immunized with pLNCX RN and pLNCX B7.2 and pLNCX 4-1BBL (titer 1:200), in comparison to a titer of 1/50 in sera of mice immunized with pLNCX RN and pLNCX B7.1 and pLNCX 4-1BBL. The in vivo effect of DNA vaccines is likely to be due to transfection of both APC and non-APC. We rationalized that antigen presentation would be optimized if the same cells were simultaneously transfected by both costimulatory molecules and we constructed a second generation of DNA vaccines using pMG vector that can accept 2 genes. Plasmids encoding CD80 and 4-1BBl (pMG B7.1/4-1BBL), and CD86 and 4-1BBl (pMG B7.2/4-1BBL) were combined to pLNCX RN. To further optimize vaccination, we also tested which DNA concentration (30ug, 10ug and 1ug) and which route of injection (ID, IM, SC or IP) induce the best expression of DNA vaccine-encoded proteins in the draining lymph nodes. We extracted RNA from inguinal lymph nodes 2 days after DNA vaccine injection, and by RT-PCR we detected the stronger transcription of Rat Neu after ID injection. By immunohistology, we demonstrated that Rat Neu, CD86 and 4-1BB ligand proteins are expressed in lymph nodes of immunized mice. Flow cytometry showed an increase of 20% of the B cell population from spleens of mice receiving Rat Neu and costimulatory molecules, as compared to mice immunized with Rat Neu alone. Furthermore, combinations of pLNCX RN with pMG B7.2/4-1BBL induce a 3 fold greater titer of HER2 specific antibodies than the first generation vaccine. We cloned HER2 high expressing tumor cells, NTT-2K, from NTT-98, a tumor cell line derived from spontaneous tumor arising in neuTg mice. NTT-2K cell growth in soft agar was inhibited in presence of sera from immunized animals. Protection assays showed that immunization with pLNCX RN and pMG B7.2/4-1BBL consistently delayed tumor development with 2 weeks following challenge with fresh tumor cells, in comparison to all other groups where no significant protection was observed. Finally, incubation of NTT-2K cells with sera from mice immunized with pLNCX RN alone increased Rat Neu phosphorylation of 5-fold (DLU), in comparison to an increase of 10 fold when combined to pMG B7.1/4-1BBL or pMG B7.2/4-1BBL.

196. Delivery of Plasmid DNA to the Skin using In Vivo Electroporation Richard Heller*†‡§, Mark Jaroszeski*†‡, Loree Heller*‡, M. Lee Lucas*§, Carlos Pottinger*†‡, Richard Gilbert*‡¶ *University of South Florida, Tampa, FL †Department of Surgery, ‡Center for Molecular Delivery §Dept of Med. Microbiology and Immunology ¶College of Engineering A key issue in the development of effective gene therapy protocols is the development of appropriate delivery methods. Safe, effective and efficient delivery of genes is paramount for the use of gene therapy to treat human diseases. Electroporation enhanced delivery of plasmid DNA is a strong candidate to meet this delivery criteria. In addition, it is also complementary to skin which is an attractive target for gene delivery because of its accessibility. Electroporation is a physical phenomena that temporarily permeabilizes cell membranes. When membranes are in a permeabilized state it is possible for molecules that do not normally pass through the membrane to gain intracellular access. Electroporation has been used in vitroto deliver genes to cells. In addition, in vivo electroporation has been used clinically to load chemotherapeutic agents into tumor cells. In a previous study, we examined the use of electroporation to deliver plasmid DNA to the skin to confirm that localized delivery can also result in increased serum levels of a specific protein. This current study was designed to examine various electroporation parameters including electrode configuration, pulse width, field strength, etc., to determine conditions that would yield optimal expression levels. The work was performed using a plasmid encoding luciferase. Intradermal injection of the luciferase plasmid alone re-

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sulted in low levels of expression. However, when combined with electroporation, expression was significantly higher. In addition, there was a marked difference in expression when comparing low voltage, long pulses to high voltage short pulses. The results presented here demonstrate that electroporation can be used to augment the efficiency of direct injection of plasmid DNA to skin. (Supported by research grant from the NIH - R21 DK055588 also supported by the Center for Molecular Delivery, Univ. of South Florida).

197. Validation of Analytical Methods for Clinical Grade Plasmid DNA Roy Musil*†‡, Melissa Rosness*†‡, Kathleen Pham*†‡, Richard Hancock*†‡, Magda Marquet*†‡ *Althea Technologies Inc. †3550 General Atomics Court, Building 2 ‡San Diego, CA, 92121 For gene therapy pre-clinical trials and early stage clinical trials, qualitative quality control assays have generally been sufficient for release of plasmid DNA. To proceed into advanced clinical trials and ultimately gain regulatory approval the pharmaceutical development of gene therapy products will have to meet the requirements for cGMP (current good manufacturing practices) production. While the “c” ostensibly stands for current, when actually following the spirit and intent of the FDA guidelines, the “c” represents control of the process and characterization of the product. There are two distinct components of cGMP, comprising of both the production and quality control operations. Production control is concerned with manufacturing. This includes the suitability of facility and staff for the manufacture of product, development of standard operating procedures and record keeping. Quality control is concerned with sampling, specifications, testing and with documentation and release procedures ensuring satisfactory quality of the final product. Appropriate analytical assays must be developed for both the in-process monitoring of the production cycles as well as for final quality control release criteria. The FDA’s “points to consider” lists some of the tests needed to confirm purity, identity, safety and potency of plasmid DNA. Measuring the relative purity and concentration of plasmid DNA by agarose gel electrophoresis or by HPLC is only a small part of the battery of analytical measurements necessary to confirm product quality. All assays must be fully validated. Most assays which have been developed for the quality control of recombinant protein drug substances need only slight adaptation for the quality control of plasmid DNA production. However, the most challenging assays in terms of unique or specific analytical tests for plasmid DNA bulk product are the measurements of protein (antigen), non-plasmid DNA and RNA trace contaminants. Documentation and validation of these assays must adhere to cGMP guidelines. Because of the theoretical possibility of integration, host cell contamination of the plasmid preparation is of particular concern. We have developed a rapid, validated, quantitative PCR assay to determine the amount of E.coli host chromosome DNA within a plasmid preparation.

198. Development of a Clinically Relevant Electroporation System for Implementation of EPO Gene Therapy Maida de las Alas*, Drew Hannaman*, Barry Ellefsen*, Gerald Nakamura*, Michael Sawdey†, Jukka Hartikka†, Robert Bernard* *Ichor Medical Systems †Vical Incorporated The safe and efficient delivery of genes in vivo has been a significant hurdle in the evolution of gene therapy. Administration of viral vectors can result in high expression levels but these vectors have safety and manufacturing issues. Non-viral plasmid DNA (pDNA) is safer and easier to manufacture, though current delivery systems have problems achieving sufficient expression levels, especially in larger animals. MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

PHYSICAL METHOD BASED GENE THERAPY Recent studies have shown that the use of in vivo electroporation in small animals can facilitate delivery of pDNA to dramatically increase protein expression in various tissues. Ichor’s patented TriGrid electroporation system is comprised of a series of needle electrodes which can be integrated to form arrays of different geometries. This flexible design allows treatment to be applied to appropriate volumes of tissue, while maintaining homogenous electric field propagation. Most importantly, this wellcharacterized system allows treatment protocols developed in small animals to be accurately scaled for clinical use. Initial studies with Balb/c mice injected in the tibialis muscle with VR2901 (10␮g/40␮l), an optimized expression plasmid encoding murine erythropoietin (mEPO), showed a greater magnitude of increase, less variability, and longer duration of elevated hematocrit with electroporation versus direct injection alone. A significant increase in serum mEPO was also observed in electroporated groups, as reported by others using “non-optimized” plasmids. However, relative to previous reports, we observed higher elevated serum mEPO levels from direct injection groups. Thus, the use of an optimized plasmid increases expression as a result of direct injection, decreasing the difference in expression observed between direct injection and electroporation. However, there is no significant effect of an optimized expression plasmid versus a non-optimized one, with electroporation. It also appears that increasing the electroporated tissue volume significantly affects the magnitude and duration of expression. This suggests that the expandable nature of this electroporation system may be beneficial in clinical administration. As a result, we sought to investigate the clinical feasibility of electroporation-mediated intramuscular delivery of pDNA encoding a therapeutic protein using our system. Large injection volumes have been suggested to help facilitate the uptake of pDNA due to increased hydrostatic pressure. On a mass basis, the typical injection volume of 40-50␮l in a mouse would translate to an injection volume of well-over 100cc in a human. We thus decreased this volume to a more clinically relevant one and performed these experiments in Fischer rats. We injected 10␮g of VR2978, an optimized plasmid encoding human EPO (hEPO), in a 10␮l volume in the quadriceps, and found an almost 60-fold increase in serum hEPO levels over direct injection. This volume in a human (2-3cc) would be more clinically relevant than comparable volumes used in mouse models. Thus, by overcoming injection volume limitations present in larger species, and enhancing the delivery and expression of small amounts of pDNA, electroporation may allow for the clinical implementation of plasmid DNA -based gene therapy. In addition, the expandability the TriGrid electroporation system would further the clinical feasibility of electroporation by providing the ability to scale up from small animals to humans.

simultaneously. Basic research and functional screening would be greatly facilitated if the initial PCR products were transcriptionally active. We have, therefore, produced a kit that allows transcriptionally active PCR (TAP) fragments to be generated from any gene of interest in 2 sequential PCR reactions. TAP fragments can be transfected into cultured cells or injected into animals resulting in expression levels comparable to supercoiled plasmids encoding the same proteins. Moreover, when TAP fragments encoding immunogenic antigens are injected into animals, antibody titers comparable to those induced by supercoiled plasmids are generated. One of the most difficult tasks in developing a DNA vaccine is the identification of the antigen that will stimulate the most effective immune response against the pathogen, particularly when the genome of the organism is large. TAP fragments may be particularly useful for scanning the genomes of complex microorganisms to identify immunogenic and protective vaccine antigens. When the number of antigens is small, like the influenza virus which encodes 11 genes, each open reading frame can be cloned and screened for immunogenic activity as a DNA vaccine in a relatively efficient manner. But when the number of antigens is in the hundreds or thousands, conventional cloning methods are too laborious and time consuming. Plasmodium falciparum, the parasite responsible for malaria, is predicted to encode more than 5,000 proteins, each of which is a potential antigen for a DNA vaccine. Two of its chromosomes have been completely sequenced and sequencing of the remaining 12 chromosomes is underway. The time and expense of individually cloning thousands of genes from P. falciparum into an expression vector is prohibitive, but it is feasible to produce thousands of TAP fragments encoding each of these genes. Here, we produce TAP fragments encoding 6 different malaria antigens representative of pre-erythrocytic and erythrocytic stage vaccine antigens; we characterized the TAP Express™ fragment’s capacity to induce parasite-specific and antigen-specific humoral and cellular immune responses in mice, and compared them with supercoiled plasmids encoding the same antigens. Extension of these methods to screen the whole malaria genome could lead to a comprehensive, and technically and logistically feasible screen of the entire malaria genome. It is now possible to identify the most efficacious antigens for a DNA vaccine, as well provide the foundation for establishing stage specific expression and subcellular localization and functional activity of these antigens.

199. Scanning the Malaria Parasite Genome for Immunogenic Antigens with TAP-Express™ Transcriptionally Active PCR Fragments

In vivo gene delivery in experimental arthritis using viral vectors has shown consistent gene expression. However, the limitations associated with viral delivery systems for in vivo gene delivery for chronic diseases, such as arthritis, necessitates examination of alternative gene transfer methods. We investigated the feasibility of synovial gene transfer using intra-articular injection of naked DNA. Arthritis was induced in Lewis rats by two weekly intradermal injections, the first of 0.4mg followed by the second injection of 0.1mg collagen in complete Freund’s adjuvant. The swollen ankle joints were injected with the luciferase reporter gene, pGL3-control plasmid DNA, as a single or multiple injections. In order to facilitate in vivo gene expression, the joints were also injected with naked DNA modified by attaching a polypeptide containing a nuclear localization signal (NLS). The joints were harvested 2-7 days following the gene delivery and extracts were assayed for luciferase activity. Minimal gene expression was seen in non-inflamed joints while gene expression in the inflamed joints varied with the disease progression. Rats injected 3-4 days after the onset of inflammation generally expressed the transgene better than those injected on the day of disease onset. The reporter gene expression was the highest when analyzed 24 hrs

Xiaowu Liang*, Denise Doolan†, Carlota Dobano†, Jiin Felgner*, Andy Teng*, Dawn Braun*, Philip Felgner* *Gene Therapy Systems Inc, San Diego, CA †USA Malaria Program, Naval Medical Research Center, Silver Spring, MD There is a growing list of microorganisms that have been completely sequenced. For example, Borrelia burgdorferi, clamydia, helicobacter pylori and tuberculosis encode 850, 900, 1600 and 4000 genes respectively. Straightforward, rapid and economical methods are available to obtain all of the necessary primers and generate pure preparations of each of these genes by PCR, but conventional PCR products are not transcriptionally active so they cannot be used directly in functional assays. The conventional way to create transcriptionally active genes is to clone the PCR fragments into an expression vector, transform and grow bacteria, and purify the plasmid. This is time and labor intensive, particularly when large numbers of genes need to be examined MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

200. Intra-Articular Gene Delivery Of Naked DNA To Rat Joints In Collagen-Induced Arthritis (CIA) Revati Tatake, Kyung Yu, Margaret O’Neill, Virginia Hikel, Josephine Pelletier, Thomas Noonan, Randall Barton Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT, 06877

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PHYSICAL METHOD BASED GENE THERAPY after injecting the plasmid DNA. Gene expression did not decline significantly until 72 hr after injection. However, rapid diminution in gene expression was observed after 72 hrs. No significant increase in gene expression was achieved by daily injections of 10-20 micro g DNA for three days in comparison with a single injection of 10-20 micro g DNA. The reporter gene expression was reduced when the plasmid DNA was linearized before injection. However, when circular DNA was modified by attaching a polypeptide containing NLS, a moderate improvement in gene expression was observed. In summary, significant gene expression was observed after intra-articular injection with circular plasmid DNA only in CIA rats with inflamed joints. The magnitude of gene expression was associated with the extent of inflammation in the injected joint. The observations using NLS-conjugated plasmid DNA suggest that modification of plasmid DNA prior to injection can further augment the transgene expression. These methods would be suitable for therapeutic genes requiring short-term expression, such as those expressing proteins to induce apoptosis for achieving therapeutic benefit.

201. Fast and Enzymeless Cloning of Transcriptionally Active PCR (TAP) Fragments Xiaowu Liang*, Andy Teng*, Shizhong Chen†, Philip Felgner* *Gene Therapy Systems Inc, San Diego, CA †Scripps Research Institute, La Jolla, CA Here we describe a fast and simple cloning system to transfer PCR fragments directly into a plasmid expression vector; this simple and straightforward method does not require treatment with restriction endonuclease, DNA ligase or any kind of DNA modifying enzymes. It is also not dependent on the ability of a specific DNA polymerase to add an additional A residue as required for the T/A cloning method. The method is complimentary to our TAP Express™ system, which uses nested PCR to append promoter and terminator sequences onto PCR fragments so that they become transcriptionally active and can be used directly in in vitro and in vivo transfection experiments. With the TAP Express™ system large numbers of genes can be conveniently amplified and introduced into functional assays in a single day, a task that is impractical or impossible using conventional cloning methodology. Here we show that when the common sequences on both the 5⬘ and 3⬘ ends of the TAP Express™ fragment are complimentary with terminal sequences in a linearized empty vector, and the fragment and linearized vector are electroporated together into bacteria, they recombine resulting in a new expression vector with the fragment directionally inserted. This new cloning approach is a companion to our TAP Express™ system in that once a gene of desired function is identified, its corresponding TAP fragment can be directly transformed into a bacterial host where the PCR fragment will be converted into a plasmid expression vector through homologous recombination. In principle this approach can be applied generally as an alternative to conventional cloning methods. Other potential applications of this enzymeless cloning approach will also be described.

202. Enhancement of Electroporation-Mediated Gene Expression In Muscle by Enzymatic Modulation of Extracellular Matrix Kenneth Liang, Leaf Huang Center for Pharmacogenetics School of Pharmacy, University of Pittsburgh Gene delivery to skeletal muscle is a promising method for the treatment of muscular diseases as well as for the production of therapeutic proteins. Electroporation has been proved to be an efficient method for gene delivery to muscle. In this study, we report a significant enhancement of electroporation-mediated gene expression in skeletal muscle by injecting plasmid DNA mixed with hyaluronidase before the application of electroporation. Luciferase gene expression was about 10 fold higher in mice injected with DNA and hyaluronidase, compared with mice in-

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jected with DNA alone. Hyaluronic acid showed no effect on promoting gene expression. Moreover, the enhancement of gene expression required enzymatic activity, because co-injection of DNA with denatured hyaluronidase or bovine serum albumin had no effect on gene expression. More importantly, the area of the muscle that expressed transgene product was increased very significantly in mice treated with hyaluronidase. More than 40% of muscle fibers expressed b-galactosidase when mice were coinjected with a plasmid containing LacZ gene and hyaluronidase. On the other hand, less than 5% of muscle fibers expressed b-galactosidase in mice injected with plasmid alone. Supported by NIH grant AR45925 and by Muscular Dystrophy Association of America.

203. The SP1017 formulation improves DNA vaccines against melanona by increasing gene expression and recruitment of immature dendritic cells Pierre Lemieux *, Nadia Gue´rin*, Alexander Kabanov†, Patricia Smith*, Valery Alakhov* *Supratek Pharma Inc., 531 Blvd. des Prairies, building 18, Laval, Que´bec, Canada H7V 1B7 †College of Pharmacy, Dept. of Pharmaceutical Sciences, 986025 Nebraska Medical Center, Omaha, NE 68198-6025 We reported recently (Lemieux, P. et al., Gene Therapy, 2000, 7, 986-991) that SP1017, a combination of 2 non-ionic block copolymers increases gene expression in skeletal muscle, dermal and tumor tissues by 10-20-fold and can be used to improve the secretion of therapeutic genes. We now report that the increase in gene expression attained with SP1017 leads to the improvement of DNA vaccines applied to cancer models. To assess modulation of DNA vaccine-specific immune responses, we have immunized C57Bl/6 mice via the i.m. route with a plasmid encoding for bgalactosidase formulated with SP1017. The results demonstrated that SP1017 significantly increased the immune responses both at the humoral and cellular levels as compared to naked DNA. Analysis of specific antibody titers demonstrated that SP1017 reduces the amount of DNA needed to produce specific immune response by 10-fold, speeds up the development of the immune response and eliminates the need for a boost immunization. Potentiating effect of SP1017 on cell-mediated immune response was demonstrated in lymphoproliferative assay. To evaluate the anticancer efficacy, the immunized mice were challenged with murine B16 melanoma cells transiently transfected with bgalactosidase. At the time of tumor challenge 15% of the mice had responded to naked DNA while 100% of the mice responded to bgalactosidase formulated with SP1017. The results demonstrated that immunization with SP1017-formulated DNA produced a strong anticancer effect and that 80% of the responding animals have rejected the tumors. Further tumor challenge experiments in a B16 melanoma model using trp-2 (tyrosinase-related protein) coding plasmid for immunization were carried out and demonstrated that the SP1017-formulated DNA produced a significant and stronger anti-tumor effect than naked trp-2. Histology analysis of muscle tissues of the animals injected with SP1017 formulated DNA revealed that this composition promotes a massive infiltration of immature dendritic cell into the injection site. These cells, when taken into a primary culture, were found to be sensitive to stimulating action of GM-CSF/IL-4, LPS and GM-CSF and responded to these stimulants by characteristic morphology changes and by high expression of major histocompatibility class II molecules. Further studies demonstrated that these dendritic cells contained a significant level of transgene product. The phenotyping studies (CD3, CD4, CD8, CD11a, CD11b, GR-1, NK1.1 and B220, MHCII markers) suggested that these cells are type 1 dendritic cells of lymphoid origin that are capable of generating antigen-specific type 1 T-cell response by inducing higher levels of Th1 cytokines, interferon g and IL-2. The infiltration of immature dendritic cells into the transgene expression site is likely associated with ability of SP1017 formulated DNA vaccine to produce strong anti-cancer effect. Thus, SP1017 may become a formulation of choice to improve multiple anticancer DNA vaccines. MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

CANCER IMMUNOTHERAPY I 204. Electroporation of naked DNA in skeletal muscle: plasmid dependence of muscle damage and effect of poloxamer 188 Michael Sawdey, Jukka Hartikka, Loretta Sukhu, Carol Buchner, Diane Hazard, Vesselina Bozoukova, Michal Margalith, Walter K. Nishioka, Carl J. Wheeler, Marston Manthorpe Vical Inc., San Diego, CA Electroporation has been reported to facilitate naked DNA gene transfer in skeletal muscle, but has also been implicated in the pathogenesis of electrical injuries. To assess the effects of electroporation on gene transfer, mouse quadriceps muscles were injected with the luciferase reporter plasmid VR1255 and electroporated with caliper electrodes. Following injection of 50 ␮g VR1255 in 50 ␮l vehicle, luciferase expression was increased 10-fold by electroporation. However, following injection of 10 ␮g VR1255 in 10 ␮l vehicle, luciferase expression was increased 70-fold by electroporation. Under optimal conditions for gene transfer, electroporation resulted in systemic elevations in creatine phosphokinase activity and the transient appearance of muscle lesions. The development of lesions was plasmid DNA-dependent, but not expression-dependent, and required the presence of plasmid in the muscle during electroporation. Co-injection of the surfactant poloxamer 188 (Pluracare F68) with VR1255 significantly reduced elevations in serum creatine phosphokinase activity following electroporation, and exhibited an additive effect on luciferase expression in muscles electroporated with 2-needle electrodes. Moreover, co-injection of poloxamer 188 increased luciferase expression 3-fold in non-electroporated muscles. Poloxamer 188 thus holds promise for improving the safety and efficacy of electroporation, and otherwise represents an excipient useful for enhancing intramuscular expression of naked DNA vaccines and therapeutics

205. Phase III comparison of intramuscular delivery of ANG1 (a Vascular Endothelial Growth Factor containing plasmid) with placebo in diabetic patients with critical limb ischaemia Yoka H. Kusumanto*, Nanno H. Mulder*, Robin P. F. Dullaart†, Jan J.A.M. van den Dungen‡, Rijk O.B. Gans§, Han H. van der Hoeven¶, Anneke M.M. Hooymans㛳, Willem J. Sluiter§, Andries J. Smit§, Rene A. Tio**, Geke A. P. Hospers*, Geke A. P. Hospers* *Internal oncology, University Hospital Groningen, the Netherlands †Endocrinology, University of Groningen, the Netherlands ‡Department of surgery, University of Groningen, the Netherlands §Department of internal Medicine, University of Groningen, the Netherlands ¶Department of Neurology, University of Groningen, the Netherlands 㛳Department of Ophtalmolgy, University of Groningen, the Netherlands **Department of Cardiology, Universitiy of Groningen, the Netherlands Object of study: Long-term clinical improvement of perfusion is sofar not possible for patients with critical limb ischaemia in whom surgical or percutaneous revascularisation is not feasible or have previously failed. This is especially true in diabetic patients where macrovascular occlusions show a predilection for distal vessels and vascular disease is characterized by microvascular dysfunction. Preclinical and recent clinical phase I/II findings suggest that intramuscular delivery of a plasmid which encodes for the Vascular Endothelial Growth Factor (VEGF) gene can promote the development of supplemental collateral blood vessels in ischaemic limbs. Our objective is to determine the effect of intramuscular administration of ANG1 (a Vascular Endothelial Growth Factor gene containing plasmid) on clinical and physiological parameters of critical limb ischaemia compared to the standard treatment in diabetes mellitus patients. Materials and methods: The present study is a double blind, placebo controlled, phase III study. Sixty patients will be included in the study. An interim analysis will be performed after treatment of 30 patients. Patients will receive a total dose of 4000 ug ANG1 or MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

placebo (saline) directly into the femoral and calf muscles of the ischaemic limb. Primary endpoints are limb survival at 100 days and an improvement of ankle brachial index of ⬎15%. Results: Twenty patients have been included in the present study since February 2000. Thirteen patients, 12 with ischaemic ulcers or gangrene and 1 with isolated rest pain, have concluded the follow-up period of 100 days. Of these patients 6 improved. Ischaemic ulcers or gangrene healed or improved markedly in three patients. Two patients experienced a regression of rest pain. In three patients hemodynamic improvement was significant (i.e. increase in toe/ brachial or ankle/brachial index of ⬎15%). Seven patients showed no response: of these 3 patients required a lower limb amputation. No substantial adverse effects have been seen in the twenty patients treated, especially no deteriortion of retinopathy. Complications were limited to transient lower extremity edema in three patients and teleangiectasia in one patient. Conclusion: Intramuscular delivery of ANG1 is acceptable and feasible in diabetic mellitus patients with critical limb ischaemia.

CANCER IMMUNOTHERAPY I 206. Dendritic Cells Transduced With HSV-1 Amplicon Vectors Expressing Model Tumor Antigens Generate Protective Tumor Immunity In Mice Richard A. Willis*†, William J. Bowers*‡§, Michael J. Turner*†, Terrence L. Fisher*†, C.Siddiq Abdul-Alim*¶, Darlene F. Howard*§, Howard J. Federoff*‡§, Edith M. Lord*†㛳, John G. Frelinger*†㛳 *University of Rochester †Department of Microbiology and Immunology ‡Department of Neurology §Center for Aging and Developmental Biology ¶Department of Biochemistry and Biophysics 㛳Cancer Center There is currently much interest in generating cytotoxic T cell (CTL) responses against tumor antigens as a therapy for cancer. Herpes simplex virus-1 (HSV-1) amplicons are plasmid-based vectors that are enclosed in HSV-1 capsids. Benefits of HSV-1 amplicons include the lack of viral coding sequences, the packaging of multiple copies of the amplicon into a single capsid resulting in an amplification of transgene expression, and the ability to transduce cells with multiple genes simultaneously. We have constructed helper virus-free HSV-1 amplicon vectors that encode the model tumor antigen ovalbumin (HSV-OVA), and human prostate-specific antigen (HSVPSA). Infection of dendritic cells (DCs), a potent form of antigenpresenting cell, with HSV-OVA or HSV-PSA and coculture with CTL hybridomas results in specific activation, indicating that transduced DCs process the tumor antigens for class I MHC presentation to CTL. Mice immunized with HSV-OVA or HSV-PSA-infected DCs generate a specific CTL response that can be detected in vitro by a 51 Cr-release assay. Mice immunized with HSV-PSA-transduced DCs are protected from challenge with PSA-expressing tumor cells. These results indicate that DCs infected with HSV-1 amplicons may provide a tool for investigation of CTL activation by DCs and a new modality for immunotherapy of cancer. This work was funded by NIH grants CA70218, CA28332, R21DK53160 and R01NS36420.

207. Comparative analysis of IFN-␥, B7.1 and antisense TGF-␤ gene transfer on the tumorigenicity of a poorly immunogenic metastatic mammary carcinoma E.T. Akporiaye*, R.S. Wu*, J.J. Kobie*, T.C. Fong† *Department of Microbiology and Immunology, University of Arizona †RPR Gencell The progression and spread of cancer have been attributed in part to immune evasion strategies which include lack of co-stimulation, down-regulation of cell surface MHC molecules and secretion of immunosuppressive factors such as transforming growth factor-␤ (TGF-␤). Gene therapeutic approaches to counter these mecha-

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