489. Further Studies of the Most Efficient Vector Systems for Gene Transduction into Human Dendritic Cells Line (im-NMD)

RNA VIRUS VECTORS: GENE TRANSFER, VECTOR PRODUCTION 489. Further Studies of the Most Efficient Vector Systems for Gene Transduction into Human Dendritic Cells Line (im-NMD) Eigo Sato,1,2 Masanari Kato,3 Yuko Hara,1 Hua Yan,1,4 Tohko Miyagi,1 Xiao-Kang Li,1 Wataru Sugiura,4 Naoki Yamamoto,4 Kenichi Teramoto,2 Shigeki Arii,2 Hiromitsu Kimura.1 1 Research Surgery, Collaborative Research, National Research Center for Child Health, Tokyo, Japan; 2Surgery, Tokyo Medical and Dental University, Tokyo, Japan; 3Pharmaceutical Business Development, Ishihara Sangyo Kaisha, Ltd, Kusatu, SG, Japan; 4 Aids Research Center, National Infectious Disease, Tokyo, Japan. It has become evident that the specialized antigen presenting cells (APC), dendritic cells (DC), play a pivotal role in initiating a primary immune response. Furthermore it has been reported that several systems including Adenoviral vector, HVJ-related vector, and electroporation, are able to transduce the gene into DC and are able to modify the function of DC in mouse and human. However, our previous study demonstrated this is not the case with rat DC. To our best knowledge, there has been no direct evidence to support that currently-used vector systems are able to effectively transduce the gene into DC. Inasmuch as most, if not all, DC or DC-related gene transfer studies appeared to be performed by employing heterogeneous cell population, it is important to determine the extent of gene transduction into bona fide DC. In this study, employing recently established human DC cell line called im-NMD and enhanced green fluorescence protein (EGFP : Clontec-C1), we further examined a relative efficiency of each vector system into one of the DC lineage cell line. We provide further evidence that none of the currentlyused vector systems are able to sufficiently transfer the gene into human DC cell line. In particular when the im-NMD is so driven by several cytokine combinations such as Flt3/Flk2 ligand and IL-6, and/or IL-4 and TNFa that they became mature DC phenotypes, it is indeed the case. Nevertheless, the most efficient transduction of the EGFP gene was observed under a long-term culture of im-NMD cell line. Hence the successful gene transfer was observed after one week culturing only with HIV-based lentiviral vector system. It should be noted that HVJ vector system that contained FITC-tagged random primers were indeed incorporated into the im-NMD. Thus it suggested that there are mechanisms that interfere the exogenous gene expression in bona fide DC.

490. Lentiviral-Mediated Gene Transfer to Cells of the Vasculature Ciara A. O’Shea,1 Robert J. Nelson,1 Padraig Strappe,1 Timothy O’Brien.1 1 REMEDI, NUI GALWAY, Galway, Ireland. Endothelial cells (EC) and smooth muscle cells (SMC) constitute the principal cellular components in both arteries and veins and play a key role in the pathogenesis of vascular diseases such as atherosclerosis and restenosis. Denudation of the endothelial layer and intimal hyperplasia development caused by the proliferation of phenotypically modified SMCs are typical clinical manifestations of such diseases. Self-inactivating third generation lentiviral vectors have previously been demonstrated as having excellant potential as tools for effective gene delivery. In this study, we have undertaken a quantitative analysis of Baculo glycoprotein (gp-64) pseudotyped lentiviral mediated reporter gene delivery to both human aortic SMCs and ECs. pWPT-GFP Baculo gp64 was produced by standard four-plasmid calcium phosphate transfection, concentrated by ultracentrifugation and quantified by

S190

p24 HIV-1 ELISA. Cells were cultured in vitro and exposed to viral vector for varying periods of time to mimic in vivo exposure in a carotid artery luminal dwell model. Transduction efficiency was quantified by FACS analysis of GFP positive cells. At the clinically relevant time point of 10min, preliminary results demonstrate a SMC transduction efficiency of 22.6% using 1.7x107IU. This increased to 26.3% following 20min exposure time. In comparison, ECs appear to have much lower transduction efficiency with this pseudotype, demonstrating only 6.8% GFP positive cells at 10mins under identical experimental conditions, and rising to 13.04% at 20min. The potential for alternative envelopes to be used in the vasculature are being investigated at present.

491. Development of Fast and Efficient Methods for the Purification and Concentration of Lentiviral Vectors Rachel M. Koldej,1,2 Donald S. Anson.1,2,3,4 Genetic Medicine, Chlidren, Youth and Women’s Health Service, Adelaide, South Australia, Australia; 2Paediatrics, University of Adelaide, Adelaide, South Australia, Australia; 3Biotechnology, Flinders University of South Australia, Adelaide, South Australia, Australia; 4Pharmacy & Medical Sciences, University of South Australia, Adelaide, South Australia, Australia. 1

Lentiviral vectors are proving to be promising tools for gene therapy. However, the relatively low titres of these vectors mean that they must be substantially concentrated before use. In addition, as with any pharmaceutical product, the final preparation must have a high and definable purity. Therefore, we are developing methods to concentrate and purify virus preparations. To facilitate processing, virus was collected in serum free medium, then clarified by low speed centrifugation and 0.45 µm filtration. As an initial purification step, several ultrafiltration systems with different molecular weight (Mw) cut-offs were evaluated and a hollow fibre system with a 750 kDa Mw cut-off was chosen as the most appropriate. Systems with membranes having a larger Mw cut-off allowed significant amounts of virus particles to pass through, while membranes with smaller Mw cut-offs resulted in slower processing and would presumably also be less effective at removing contaminants. Ultrafiltration allowed the clarified cell culture supernatant to be concentrated 20-fold in 2-3 hours, with virus recoveries of approximately 50% and a greater than 10-fold reduction of total protein. Ultracentrifugation was then used to pellet the virus, allowing it to be resuspended in a small volume (approximately 1000th of the starting volume) of the buffer of choice. Recovery of virus was again about 50% and total protein was reduced a further 10-fold. The combination of ultrafiltration and ultracentrifugation has resulted in the rapid concentration and purification of large volumes (over 3 litres) of virus supernatant, with overall recoveries of >25% of infectious virus and a reduction to <2% of the initial amount of protein per infectious-unit. This approach was also rapid: the overall processing time from collection of viral supernatant to resuspension of the final virus preparation was <8 hours. However, SDS-PAGE analysis suggests the final material is still not 100% pure. We are now exploring the effects of varying ultrafiltration and ultracentrifugation parameters with the aim of raising the overall viable virus particle recovery to at least 50%. In addition, we are investigating the addition of an anion exchange chromatography step to increase virus recovery and simultaneously increase the purity of the final virus preparation.

Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy