538 Cytoskeletal Involvement in the Albumin Facilitated Lipofection Gene Delivery Strategy Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������© ����������� �!����� ����"� ��������[.]
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 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 with a decrease in transfection efficiency Approximately 14% of the [125I]HSA in the optimal formulation was in DC-HSA-DNA ternary complexes as shown by Sepharose 4B column chromatography Measured by light scattering, the size of the transfection complexes in the optimal formulation was found to be 2-2.5 times that of DC as well as DC plus DNA complexes Transmission electron microscopy examination of cells transfected with a formulation prepared with [20 nm-gold]albumin, DC, and [10 nm-gold]DNA showed DNA in an extended conformation at the cell surface and co-localization of albumin and DNA intracellularly The data suggest that albumin prevents DNA condensation by the liposome, co-transports with the DNA, and facilitates nuclear targeting of the DNA Despite its extended nature in the transfection complexes, the DNA was resistant to DNase treatment To examine the mechanism of endocytosis and intracellular trafficking of the transfection complexes, various inhibitors were utilized Pretreatment with chlorpromazine and excess HSA caused a 30% and 25% decrease of transfection efficiency, respectively, whereas no apparent effect was observed for filipin complex The results suggest involvement of an albumin receptor and clathrincoated pits, but not caveolae in endocytosis of the transfection complexes Treatment with the three classes of microtubule modifying-agents, such as colchicine, paclitaxel, and vinblastine enhanced the transfection efficiency 2-, 2-, and 1.5-fold, respectively However, an actin inhibitor, cytochalasin B, caused 60% inhibition We conclude that in our gene delivery strategy, albumin and DNA are co-transported to the nucleus via an actin-dependent and microtubule-independent mechanism Incorporation of cytoskeletal modulating agents in the albumin-facilitated lipofection gene delivery strategy should further the potential utilization of this non-viral vector for in vivo gene therapy 539 Chondrogenic Potential of Direct Plasmid Gene Delivery in Three Dimensional Culture Alicia L Bertone, Rhonda Schreiber, Stephen B Trippel Veterinary Clinical Sciences, The Ohio State University, Columbus, OH, United States; 2Selective Genetics, Inc, San Diego, CA, United States; 3Orthopedics, University of Indiana, Indianapolis, IN, United States Several morphogens have the ability to enhance cellular proliferation (ie, FGF-2) and expression of proteoglycan [PG] and type-2 collagen (ie, IGF-1 and BMP-2) in chondrocytes Delivery of these genes can be therapeutic Nonviral (vs viral) gene delivery offers advantages, but suffers from lower transfection Our study (1) optimized direct DNA transfer in a 3-D cell system that can be implanted in vivo using plasmid DNA and (2) evaluated hFGF, hIGF1 and hBMP2 to promote chondrocyte proliferation and expression of matrix using a 3-D collagen suspension Chondrocytes from the knee joint of calves were suspended in constructs of known cell density with the desired DNA and lipid [Lipofectamine2000] concentration in type-1 collagen gel and cultured for 24 days Optimal cell density [0.5, 1, and x 106 cells/well], pDNA/well [0, 0.9, 1.86, 4.65, 9.3, 18.6, 37.2, and 74.4 ug DNA], and lipid reagent [2:1, 3:1 and 6:1 DNA/Lipid ratio] for transfection were quantified with plucerifase [pg/ml] and %GFP+ cells ugdna 0.9 4.65 9.3 18.6 37.2 %GFP+cells for each of lipid/dna ratios Lipo/dna ratio %pos Lipo/dna ratio %pos Lipo/dna ratio %pos 0 3:1 6:1 0 3:1 1.6 6:1 2.5 0 3:1 10.8 6:1 3.3 0 3:1 13.3 6:1 8.1 0 3:1 50.5 6;1 Based on the results of the optimization studies, x 104 cells/ well, 37.2 ug DNA, and a 3:1 lipid/DNA ratio (111.6ug lipid) were used in the following morphogen groups: (1) No DNA, No lipid Control [NoL/NoD]; 2) No DNA, lipid Control [L/NoD]; 3) S210 pLuciferase, lipid; 5) phFGF2, lipid; 6) phIGF1,lipid; and 7)phBMP2, lipid Outcome measures included cell viability, tranfection efficiency (%), cell proliferation (doubling time), protein expression (pluciferase), proteoglycan production (Alcian blue stain intensity) and morphology (Eosin stain) Gene transfer and expression occurred only when supplemented with lipid agent Growth factors promoted the health of chondrocytes in this 3-D culture system hIGF and HBMP2 promoted proteoglycan production to the greatest extent and had the maximal morphology scores FGF and BMP2 promoted cell health and growth to the greatest extent based on appearance, cell number and viability Plasmid delivery of morphogens enhanced cell proliferation, viability in the presence of lipid agent, and maintenance of chondrogenic phenotype Group Doubling (hrs) Day 7- % Viability Alcian stain Morph Score NoL/NoD 96 100 Normal L/NoD 36.1 23 0 Crenated L/pLuc 22.7 78.2 1+ L/pFGF 20.6 74.2 1.5+ L/pIGF 23.3 57.5 4+ L/pBMP2 21.7 84.5 4+ Lipid agent at the concentrations necessary to support acceptable direct plasmid gene transfer to chondrocytes (~15%) is biocompatible with the cells in the shorter term (up to days), but prolonged exposure (7 days) resulted in altered morphology and cell death Transfection efficiency in this 3-D cell suspension produced expression comparable to other lipid based methods Relevant effectiveness in vivo may be achieved in 3-D cellular constructs without vector delivery Acknowledgement: CH Evans for consultation/facilities 540 A Novel Analogue of DOPE Assists Formulation of Lower-Charged, Highly Transfecting Cationic Liposomes for Gene Delivery Steven Fletcher, Michael R Jorgensen, Kostas Kostarelos, Andrew D Miller Imperial College Genetic Therapies Centre, Department of Chemistry, Imperial College London, London, United Kingdom Towards enhancing the in vivo gene transfer efficiency of cationic liposomes, it is necessary to reduce the magnitude of the cationic charge of the liposome-based systems, without impairing transfection To this end, we have prepared a series of monoacetylenic analogues of the neutral, helper lipid dioleoyl-L-αphosphatidylethanolamine (DOPE) in the belief that these structural changes should raise the Lα / HII phase transition temperature, Th, above that of DOPE (10 °C) Provided Th does not exceed the physiological temperature, then not only should the fusogenicity of the original DOPE molecule be maintained but the increased stability at lower temperatures should enable the preparation of lowercharged, yet highly transfecting, cationic liposomes through enhanced DOPE-analogue content Through 31P NMR spectroscopy, we report the phase behaviour of these analogues As predicted, all Lα / HII phase transition temperatures are greater than that of DOPE, but only one analogue, namely DO(9-yne)PE, adopts the HII phase below the physiological temperature (Th = 25 – 30 °C) The phase behaviour of this lipid, relative to DOPE, prompted us to attempt the preparation of lowercharged, cationic liposomes Within our group, cationic liposomes are generally created through mixing DOPE with our cationic lipid CDAN in a 1:1 molar ratio, then extruded and sized (typically, 100 +/- 10 nm (photon correlation spectroscopy (PCS)) Meanwhile, the reporter plasmid (pUMVC1-nt-beta-gal, 7.53 kbp) is precondensed with the adenoviral core peptide mu in a 1:0.6 (w:w) ratio, respectively Complexation of this MD species with cationic liposomes in a 1:12 (w:w) ratio then generates the LMD particle (140 +/- 10 nm (PCS)) we routinely use for in vitro transfection studies Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy ... in our gene delivery strategy, albumin and DNA are co-transported to the nucleus via an actin-dependent and microtubule-independent mechanism Incorporation of cytoskeletal modulating agents in. .. cytoskeletal modulating agents in the albumin- facilitated lipofection gene delivery strategy should further the potential utilization of this non-viral vector for in vivo gene therapy 539 Chondrogenic... nm-gold ]albumin, DC, and [10 nm-gold]DNA showed DNA in an extended conformation at the cell surface and co-localization of albumin and DNA intracellularly The data suggest that albumin prevents