Polymer binding to DNA neutralizes anionic charges on the phosphate backbone, rendering the complex immobile under electric field. Binding was generally ...
DNA VECTOROLOGY Polymer binding to DNA neutralizes anionic charges on the phosphate backbone, rendering the complex immobile under electric field. Binding was generally poor for the linear PEIs as most of the DNA migrated through the agarose matrix. Lipofectamine only showed a preferential binding for linear DNAs. PLL-PA and PLL binds equally well to all three types of DNA. Branched PEI showed a significant selectivity for both linear plasmid DNA and linear PCR product over the circular plasmid. In short, a correlation between binding affinity and DNA physical properties cannot be generalized for all polymers. Almost all of the BMSC transfected with branched PEI as polymer exhibited DNA uptake. PLL, PLL-PA and Lipofectamine2000 all induced an elevated level of DNA uptake, but at a lower level compared to branched PEI. Linear PEI was least effective as delivery vector, consistent with very low binding data. No apparent correlation between DNA uptake and the differences in molecular weight and conformation can be drawn. Polymers also did not show a clear pattern about the relative efficiency towards different DNA molecules. In conclusion, non-viral polymer vectors are capable of transferring different sizes of DNA into the cell with no effect of being either of linear or circular configuration.
531. Complexes of DNA-Cationic Liposomes with Baculovirus GP64 Envelope Fusion Protein Vesicles Demonstrate Enhanced Transfection Efficiency Ghiabe H. Guibinga,1 Shaochun Song,1 Jeanne Loring,2 Theodore Friedmann.1 1 Department of Pediatrics, UC San Diego, La Jolla, CA; 2Stem Cell Research Center, Burnham Institute, La Jolla, CA. The need for efficient and safe gene transfer reagents has led to the growing interest in a class of hybrid vectors comprising both viral and non-viral components can be associated. We have examined the transfection efficiency of DOTAP-based lipoplexes together with partially purified vesicles of the baculovirus GP64 envelope glycoprotein eihter alone or as a fusion protein of GP64 with the complement-inactivating decay accelerating factor (DAF, CD55). The addition of GP64 vesicles to lipoplexes expressing luciferase gene enhances the transient transfection efficiency in several target cells in tissue culture by up to approximately 15-fold, as measured by luciferase activity. The enhanced transfection is markedly inhibited by the addition of a GP64-neutralizing antibody. GP64mediated enhancement of DOTAP-based lipoplexes is associated with physical interaction between the DNA-liposomes with the GP64/ fusion protein, as assayed as by sucrose density gradient centrifugation. These results complement our previous demonstrations of enhanced transfection efficiency of complexes of plasmids or lipoplexes with the VSV-G glycoprotein and suggest that several kinds of viral envelope proteins facilitate the attachment, cell entry and gene transfer efficiencies of nanoparticle complexes.
532. An Inducible Fed-Batch Fermentation Process for Scale-Up and Production of DNA Vaccines and Gene Medicines Aaron E. Carnes,1 James A. Williams,1 Clague P. Hodgson.1 1 Research and Development, Nature Technology Corporation, Lincoln, NE. It is essential to devise industrial processes whereby plasmid DNA can be manufactured to meet the quality, economy, and scale requirements projected for future gene medicine and DNA vaccine
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production. To date, most plasmid fermentation media and processes result in low yields, imposing a cost and purity burden on commercialization of plasmid DNA production. Here we report the development of an inducible fed-batch fermentation process that dramatically increases volumetric yield and specific plasmid yield with respect to cell mass, while maintaining or enhancing plasmid integrity. Additionally, this process is well suited for producing unstable plasmids. This inducible process utilizes commercially available media that we designed specifically for plasmid production. The process consists of an initial biomass accumulation phase, followed by a plasmid accumulation phase. The plasmid is stably maintained at low levels during a period of nutrient restricted growth and reduced temperature (30°C), and then the temperature is increased (37-42°C) to induce plasmid amplification. Typically, the specific plasmid yield increases over a period of up to 8 hours following temperature up-shift. A set of ten replicate fermentations producing a DNA vaccine plasmid was performed. The total process lasted 38.5 hours, with a 7 hour induction time. The overall plasmid yield averaged 760 mg/L, and the average specific plasmid yield was 8.5 mg/L/OD600. A total DNA analysis by AGE of total cell lysates indicated that plasmid DNA accounted for the majority of the total DNA in the cells at harvest. A high degree of process robustness and reproducibility has been demonstrated with many different plasmids and with multiple batches of a single plasmid. Fed-batch fermentation productivity exceeding 1000 mg plasmid DNA/L fermentation media has been obtained with pUC origin containing plasmids (see figure). This five to ten fold increase in plasmid yield dramatically decreases plasmid manufacturing costs, and improves the effectiveness of downstream purification by reducing the fraction of impurities.
533. Recombinant (KHG)6-FGF2 Biopolymer for Targeted Gene Transfer Arash Hatefi, Hamid Ghandehari. 1 Centre for Nanomedicine and Cellular Delivery, Pharmaceutical Sciences, University of Maryland, Baltimore, MD. INTRODUCTION Previously, we reported the biosynthesis, characterization, cellular recognition and transfection efficiency of a de novo designed targetable biopolymer, namely (KHKHKHKHKK) 6 -FGF2 [1,2]. This construct with exact sequence and length (referred to as (KH)6FGF2) contained Lysine (K) residues to allow efficient complexation with plasmid DNA, human basic Fibroblast Growth Factor (FGF2) to target cells over-expressing FGF2 receptors (FGFR), and
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
