535 Poly Propyl(Acrylic Acid) (PPAA) Functions as an Endosomal Escape Agent Synergistically Enhancing the Transfection Potency of Folate Targeted Cationic Lipid Protamine DNA (LPD) Complexes Molecular[.]
LIPID MEDIATED GENE TRANSFER Continuing work aims to quantitatively evaluate the mechanisms and kinetics of siRNA in modulating the expression of both endogenous and exogenous proteins in mammalian cells for optimized RNAi function 533 High Levels of Foreign Gene Expression in Hemophiliac Mice after Intravenous Injection of a Non-Viral Vector A T M S Hoque,1 Richard H Smith,2 Robert M Kotin,2 Basil Golding.1 Laboratory of Plasma Derivatives/Division of Hematology, Food and Drug Administration/CBER/OBRR, Bethesda, MD, United States; 2Laboratory of Biochemical Genetics, National Heart Lung and Blood Institute, NIH, Bethesda, MD, United States We are considering use of non-viral plasmid DNA gene transfer technology for treating single protein deficiency disorders such as Hemophilia A We have constructed an expression vector using adeno-associated virus type (AAV-2) backbone including internal terminal repeats (ITRs) and encoding the luciferase reporter gene In order to optimize expression of the transgene product we used several different formulations, i.e saline (pH 7.6), Hanks’ Balanced Salt solution (HBSS, pH 7.6), MirusTransIT (Mirus) , Lipofectamine 2000 (Invitrogen), Polyethylenimine (PEI; 9:1) in 20 mM HEPES (pH 7.4), PEI (9:1) in 5% glucose solution (pH 7.2), PEI (6:1) in 5% glucose solution and combination of PEI (9:1) in Lipofectamine 2000 and 5% glucose solution, to deliver the AAV-2 expression vector in vitro and in vivo First, we evaluated the ability of these formulations for transfection of plasmid DNA vector into human embryonic kidney cells (293) Our results show that the use of a formulation containing a mixture of PEI (9:1), Lipofectamine 2000 and glucose results in increased luciferase gene expression in the cells compared to other formulations Secondly, we evaluated the ability of these formulations to transfect the liver and other organs after tail vein injection in hemophiliac mice Our in vivo results show the combination of PEI (9:1), Lipofectamine 2000 and 5% glucose results in high levels of luciferase expression in the liver of the hemophiliac mice up to seven days after administration compared to other formulations We also examined the effects of the formulations used to deliver plasmid DNA on clinical chemistry and hematology values of the mice Our results show that none of these formulations, except PEI alone, had any significant effects on these parameters We conclude that the combination of PEI, Lipofectamine 2000 and 5% glucose is able to significantly enhance luciferase gene expression in vitro and in vivo with no evidence of toxicity and may be useful to facilitate expression of therapeutic gene expression as well 534 Lipid-Based hVEGF Gene Delivery to Human Pancreatic Islets Kun Cheng,1 Ajit S Narang,1 Daniel Fraga,2 Abdel-Baset Halim,2 Malak Kotb,2 Osama Gaber,2 Ram I Mahato.1 Pharmaceutical Sciences, University of Tennessee, Memphis, TN, United States; 2Surgery, University of Tennessee, Memphis, TN, United States Introduction Human islet transplantation has great potential as an effective means of treating type-I diabetes, it is still limited primarily because a large number of transplanted islets never function and islets undergo apoptosis Islet destruction following transplantation can be prevented by hVEGF, which can promote islet revascularization Co-expression of multiple genes would be an effective strategy to promote revascularization and intercept apoptosis In this study, we investigated the possible use of plasmid and adenovirus-based biscistronic vectors encoding EGFP and hVEGF for gene delivery to human islets Methods Pancreas were S208 obtained from heart-beating cadaveric multiorgan donors, perfused and digested Islets were transfected with pCMS-EGFP-hVEGF, pCMS-EGFP and pCAGGS-hVEGF after complex formation with LipofectAMINE Samples were withdrawn at days 1, and 10 post-transfection, and supernatants were analyzed for hVEGF by ELISA Islet funtion after transfection was determined by measuring secreted human insulin by ELISA In vivo function of transfected islets following transplantation of normal mice was determined by measuring plasma insulin levels Results Secreted hVEGF levels from transfected islets was significantly different for the bicistronic and corresponding monocistonic plasmids, with the level being higher for the bicistronic plasmid hVEGF expression levels did not reduce or even level off and persisted over a period of 10 days We evaluated the time profile of islet cytotoxicity after transfection and found 93% viable cells which remained almost the same at all time points To determine islet functional viability after transfection, we measured insulin release in response to glucose stimulation (Fig.1) The stimulation index of insulin secretion by the islets was calculated as a ratio of insulin secretion in response to stimulated (higher) glucose and to lower (basal) glucose concentrations SI of the bicistronic plasmid transfected islets was comparable to that of the non transfected islets Furthermore, reduction of glucose concentration back to basal level has resulted in reduction in insulin response to basal level in all cases, indicating that the islets remain dynamically responsive to glucose concentration Adequate levels of insulin release up to 10 days post-transfection and after transplantation into normal mice were also observed Conclusions We have demonstrated higher hVEGF expression after transfection of human islets with LipofectAMINE/pCMS-EGFP-hVEGF complexes Our future work will include replacing EGFP with IL-1Ra in the bicistronic plasmid, immunohistochemistry, bioactivity assays of therapeutic genes, and transfection using modified adenoviral vectors Acknowledgements We acknowledge UT and NIH for funding and James Henry for technical support References Mahato et al Molecular Therapy (2003) 7:89-100 535 Poly Propyl(Acrylic Acid) (PPAA) Functions as an Endosomal Escape Agent Synergistically Enhancing the Transfection Potency of Folate Targeted Cationic Lipid-Protamine-DNA (LPD) Complexes Ralph W Paul,1 Benjamin Dutzar,1 Pat S Stayton,2 Allan Hoffman,2 Pervin Anklesaria,1 Pierrot Harvie.1 Department of Research, Targeted Genetics Corporation, Seattle, WA, United States; 2Department of Bioengineering, University of Washington, Seattle, WA, United States After binding to target cells cationic lipid based gene delivery systems typically enter through an endosomal pathway Internalization into this degradative pathway represents one of the most crucial hurdles which must be dealt with if more efficient synthetic delivery systems are to be developed In this study, poly propyl(acrylic acid) (PPAA) a pH sensitive polymer known for its membrane disruptive capacity at endosomal pH (pH = mg PPAA/mg DNA, an optimal enhancement of in vitro transfection activity was observed Formulations containing PPAA demonstrated a shift in their zeta potential from a negative zeta potential at pH 7.4 to a positive zeta potential at pH 4.2 indicating that the predicted charge shift associated with the polymer at low pH was operational in the formulation context Incorporation of PPAA into LPD formulations enhanced the in vitro transfection of KB cells by 1-2 logs over the unmodified formulation However, incorporation of PPAA in these formulations increased the mean formulation particle diameter from ~200 nm up to 800 nm This size alteration was prevented when the LPD formulations were modified through the incorporation of DSPE-PEG5K (LPD-PEG) or DSPE-PEG5K-Folate (LPD-PEG-Folate) PPAA addition to LPD-PEG formulations enhanced transfection potency logs over unmodified LPD-PEG formulations More interestingly, a log transfection enhancement was observed for LPD-PEG-Folate formulations containing PPAA, indicating a synergistic effect between the polymer and the Folate ligand These transfection enhancements were maintained or improved upon even after incubation of these formulations for 1h incubation at 37ºC in mouse serum prior to transfection This suggests that these new formulations may be viable for in vivo application Addition of Chloroquine to transfections inhibited the observed PPAA mediated transfection enhancement This supported the possible importance of the endosomal acidification in PPAA mediated transfection enhancement Cumulatively, this data suggests that this modification of a multifunctional gene delivery system represents an additional step in the generation of synthetic delivery systems with greater transfection efficiencies 536 Role of the Toll-Like Receptor in the Acute Inflammatory Response to Nonviral Vectors Hongmei Zhao,1 I.-Huan Wu,1 Hiraoki Hemmi,2 Shizuo Akira,2 Seng H Cheng,1 Ronald K Scheule,1 Nelson S Yew.1 Gene Transfer Research, Genzyme Corporation, Framingham, MA; 2Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan We have shown previously that cationic lipid-plasmid DNA (pDNA) complexes containing pDNA vectors that have been largely depleted of CpG motifs have reduced toxicity when delivered systemically However, several CpGs remain in these vectors and the acute toxicity is not negligible, especially at higher doses of complex To determine which toxicities can or cannot be resolved by completely eliminating CpG-mediated stimulation, we measured the response to systemically delivered complexes in transgenic mice that are deficient in the Toll-like (TLR9) receptor, which is the receptor that recognizes immunostimulatory CpG motifs One day after injecting complex, the proinflammatory cytokines IL-12, IL-6, and IFN-gamma remained at near background levels in the TLR9-/mice A significant decrease in adverse hematological changes and reduced elevations of the liver enzymes ALT and AST were also observed in the TLR9-/- mice compare to normal mice, with a concomitant marked improvement in survival However, a pronounced loss of lymphocytes and platelets was still observed in the TLR9-/- mice at higher doses, similar to that seen in normal C57BL/6 mice We also measured the toxicity in normal mice of systemically delivered complexes containing non-CpG oligonucleotides or DNA fragments that are nearly devoid of CpGs Although ALT and AST levels were reduced, a loss of lymphocytes and platelets was observed akin to that seen in the TLR9-/- mice Taken together, these results suggest that signaling through the TLR9 receptor contributes to some but not all of the toxic responses associated with systemic delivery of cationic lipid-pDNA complexes The results also imply that a completely non-CpG pDNA vector Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy will have limited but nevertheless beneficial effects on decreasing liver damage and increasing the safety of systemically delivered nonviral vectors 537 Microtubule-Disrupting Agents Increase Transgene Expression in A549 Cells through the Activation of the Src and ERK Kinase Pathway Rajesh R Nair,1 Lindsay A Schwarz.1 Dept of PPS, University of Houston, Houston, TX, United States Colchicine, a microtubule-disrupting agent, has been proposed to enhance transgene expression in cells by inhibiting the endo-lysosomal fusion We found that in several different human cell lines, including A549 human lung adenocarcinoma cells, hr treatment with μM colchicine resulted in a 15-30-fold increase in transgene expression Increased transgene expression was evident even in cells not treated until 12 hrs after transfection, with μM colchicine In addition, when cells were pulsed for hr with μM colchicine and transfected after 18 hrs post-treatment, enhanced transgene expression was still observed These data suggested that the colchicine-mediated increase in transgene expression occurs through induction of a facilitating factor and not through inhibition of endo-lysosomal fusion To test whether microtubular depolymerization was critical to the activation of this factor, cells were pretreated with a microtubular stabilizer, paclitaxel, hrs before colchicine Paclitaxel pretreatment dosedependently and significantly inhibited the colchicine-induced increase in transgene expression, suggesting that microtubular disruption is pivotal for the activation of the factor facilitating the increase in transgene expression Colchicine also activates the src family kinases which, in turn, leads to the sustained activation of ERK1/2 To investigate the significance of this pathway in the colchicine-enhanced transgene expression, we treated cells with PP1, a specific src kinase inhibitor PP1 dose dependently and significantly inhibited the colchicine-induced increase in transgene expression, suggesting a role of src kinase in this pathway Further, PD98059 an inhibitor of ERK1/2, also dose dependently and significantly decreased colchicine-enhanced transgene expression The ability of PD98059 to inhibit ERK1/2 was confirmed by probing for activated ERK1/2 by Western blotting Since ERK1/2 regulate transcription of certain cellular genes, we tested if new transcription is needed for the colchicine-enhanced transgene expression Actinomycin D, an inhibitor or transcription, dose-dependently and significantly inhibited the colchicine effect In summary, colchicine appears to increase transgene expression via a signaling pathway initiated by microtubular depolymerization and mediated by activation of src and ERK kinases, which subsequently modulate transcription 538 Cytoskeletal Involvement in the AlbuminFacilitated Lipofection Gene Delivery Strategy Robert W Arpke,1 Pi-Wan Cheng.1,2 Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE; 2Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE Our lab has shown that supplementing liposome with a protein, such as transferrin, insulin, or lectins can circumvent the limitation of low transfection efficiency of liposome-based gene delivery strategies Recently, we found that human serum albumin (HSA) can enhance the lipofection efficiency 10-20 fold in a human pancreatic cancer cell line, Panc The purpose of this study is to characterize the albumin-facilitated lipofection gene delivery strategy and elucidate its gene targeting mechanism By agarose gel electrophoresis, the amount of DMRIE-Cholesterol (DC) required to fully complex μg of pCMVlacZ in the presence of 0.05 μg HSA was determined to be 5.5 μg Upon addition of increasing amounts of HSA, DNA was excluded from the complexes, corresponding S209 .. .LIPID MEDIATED GENE TRANSFER Cationic lipid: Protamine: DNA (LPD) formulations and at a specific ratio of >= mg PPAA/mg DNA, an optimal enhancement of in vitro transfection activity was observed... log transfection enhancement was observed for LPD-PEG -Folate formulations containing PPAA, indicating a synergistic effect between the polymer and the Folate ligand These transfection enhancements... Chloroquine to transfections inhibited the observed PPAA mediated transfection enhancement This supported the possible importance of the endosomal acidification in PPAA mediated transfection enhancement