plasmid DNA mediated gene transfer

MOLECULAR CONJUGATES 850. Quantitative Analysis of Non-Viral Gene Therapy in a Three-Dimensional Liver Tissue Construct Nathan C. Tedford,1,2 David W. Jackson,3 Karel Domansky,1,2 Linda G. Griffith,1,2,4 Douglas A. Lauffenburger.1,2,3,5 1 Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA; 2Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge, MA; 3 Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA; 4Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA; 5Department of Biology, Massachusetts Institute of Technology, Cambridge, MA. Successful delivery of DNA lies at the heart of gene therapy, and its feasibility in treating a number of diseases depends on the continued development of more effective gene delivery vectors. While vectors based upon recombinant viruses have shown high transfection efficiencies, they may also pose certain health risks to patients and can be difficult to target to individual cell or tissue types of interest. Non-viral vectors look to offer a safer alternative and can be engineered to more effectively treat a specific cell type, tissue, or pathology, but these vectors are still plagued with low transfection levels. Many barriers exist in the successful trafficking of these non-viral complexes to the nucleus. Current evaluations of non-viral gene delivery treatments in more clinical settings often focus on a single barrier at a time, and as a result, may not lead to an overall improvement in gene delivery. Concurrently, more quantitative or systematic in vitro experiments may not correlate well with in vivo data. A scaled up and improved three-dimensional, perfused bioreactor has been designed and built that allows for the long-term culture of primary hepatocytes. Within the microfabricated flow channels of this reactor, cells self assemble over time into tissue structures that more closely mimic hepatic morphology and phenotype than conventional two-dimensional culture systems. By studying non-viral gene delivery in this system, quantitative experiments and experimentally-driven computational models can be developed that may better describe how a vector will perform in vivo. Methodologies in density gradient electrophoresis (DGE) have been adapted to obtain greater resolution in subcellular fractionation. An experimental scheme has been developed which utilizes a newly constructed DGE device that has demonstrated proof of principle for the separation and collection of the vesicular organelles that play an important role in gene delivery. Combined with quantitative downstream assays for both the DNA plasmid and the polymer carrier, vector dynamics can now potentially be tracked at cell entry, progressive stages of vesicular trafficking and escape, and nuclear import, providing data sets which may in turn lead to more accurate and predictive mathematical models. Through a systematic iteration of quantitative experiments and computational simulations, these models will be fine-tuned for different polymer carriers administered to the hepatic tissue constructs, potentially allowing for optimization of specific vector properties and increased success of non-viral approaches.

851. Development of Brain Tumor Organ Cultures for Characterizing Polymer/Plasmid DNA Mediated Gene Transfer Nathalie Y. Toussaint, Sean Kearns, Bjorn Scheffler, Ronald Mandel, Sean M. Sullivan. 1 College of Pharmacy, Department of Pharmaceutics, University of Florida, Gainesville, FL; 2Neuroscience Science, University of Florida, Gainesville, FL. Development of effective gene therapy requires an expeditious and reproducible test system. The syngenic RG2 rat brain tumor Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy

model satisfies the criteria for a clinically relevant animal model. Pluronic block copolymer L44 was formulated with a green fluorescent protein (GFP) expression plasmid, and sterotacticly injected into rat brain tumors. GFP positive cells were observed in the tumor. Yet, the numbers of transfected cells were insufficient for a therapeutic effect. Hence, the formulation requires other components to increase gene transfer efficiency. The turn around time for obtaining results from this animal model was not amendable to screening formulations for tumor gene transfer. Cell culture using rat glioma cell lines has proven to be advantageous in screening gene transfer vectors. Conversely, due to the two-dimensional single cell layer and volume of growth media necessary for tissue culture, it is not applicable for evaluating local administration of plasmid DNA. For this reason, an organ culture was developed to optimize screening. Rat brains tumors were sliced into 300-micron sections and cultured for 7days. Tumors were observed to grow over the 7-day period ultimately taking over the brain slice. Local injection of Poloxamer formulated GFP expression plasmid was shown to transfect tumor cells in the organ culture model. Presently, the brain organ cultures are being characterized for expression of normal and brain tumor cell markers. Comparison of staining patterns for biomarkers in the organ culture and in vivo brain tumors are being used to validate the organ culture. Concomitantly, formulation conditions are being tested to optimize gene transfer to the brain tumor.

852. Development of Systemic Non-Viral Gene Delivery Systems: Cationic Nanogels Serguei V. Vinogradov,1 Alexander V. Kabanov.1 Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE.

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Development of injectable non-viral gene delivery systems as an alternative of sometimes unsafe viral gene therapy is a fast-growing area of research. However, many of the existing non-viral DNA delivery systems show poor performance in vivo. Cationic Nanogel particles have been recently used in delivery of antisense oligonucleotides. They are constructed from interpenetrated chains of branched PEI with molecular weight 25KDa (PEI25) and covalently-linked polyethylene glycol forming cationic hydrogel network with particle size of 200 nm (Figure 1). These carriers have been shown to provide an excellent nuclease protection of plasmid DNA in circulation. We report here first steps toward an improvement of transfection efficacy of Nanogel/plasmid complexes in cancer cell cultures. First, Nanogel formed compact complexes with plasmid at high excess of charged PEI amino groups over phosphates of DNA, forming spherical particles with diameter of 80 nm. These complexes were stable in dispersion and formed even smaller particles in the presence of serum. Transfection of prostate carcinoma PC-3 cells with plasmid encoding the firefly luciferase gene was dose-dependent process and very low cellular toxicity of Nanogel was observed at DNA doses 2-5 µg/ml. Transfection by the Nanogel/plasmid complex did not depend on presence of serum (10% FBS) in the medium. Second, a surface modification of Nanogel with human transferrin via non-degradable linker demonstrated 3040-fold increase of transfection efficacy of corresponding hTfNanogel/plasmid complexes. hTf-Nanogel having 5-7% of attached protein by weight was found the most active and did not loose useful properties. hTf-Nanogel and plasmid formed complex of 100 nm in diameter, which was stable in the presence of serum. This binding of hTf-Nanogel was receptor-mediated and suppressed by the presence of free transferrin in the medium. Third, preformulation of DNA with protamine (PA) in quantity of 10% of total DNA phosphates, before formation of complex with Nanogel additionally enhanced the efficacy of transfection approximately 2-fold. We hypothesize that PA is left bound to the plasmid following its release from Nanogel into cytosol and provides protective function S323