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LIPID MEDIATED GENE TRANSFER widely: viral and non-viral The advantages of non-viral over viral vector are safer, less immunogenic, easy to handle and low cost However, the drawbacks for current commercial non-viral vectors are well recognized as high cytotoxicity and low transfection efficiency A novel gene transfer technology related to lipid-DNA complexes have been developed through a rational molecular design based on cell endosome disruption and control-release mechanisms The internalization of DNA/lipid complexes is believed through endocytosis pathway Once inside the cells, the lipid-DNA complexes are trapped inside endosome, where they tend to be retained at acidic condition (pH value is around rather than 7.4 as the physiological condition) The Vaxim’s proprietary lipids are biodegradable inside the endosome under this acidic condition As the result of this degradation, DNA is slowly dissociated from the complexes It is believed that the fragments of degraded lipids are able to buffer and eventually destabilize the endosomes This will increase the chance of the DNA escaping and crossing nuclear membrane to increase the gene expression The formulations have a very low cytotoxicity (over 90% cell survival rate) and high transfection efficiency (over 90% cells are transfected in some cell lines) The results of transfecting various cell lines for recombinant protein productions will be reported The formulations have also been used for establishing stably transfected tumor cell lines for continuous recombinant protein productions as well as been used as lipid-DNA vaccine for cancer immunotherapy applications 566 Trojene™: A Novel Cationic Liposome Formulation with Minimum Handling Features and Serum Compatibility for Non-Viral Gene Delivery In Vitro Annabelle Caussin,1 Sebastien Spagnou,1 Michael Keller,1 Michael Jorgensen,1 Kristina Ulrich,1 Andrew D Miller,2 Eric Perouzel.1 IC-Vec Ltd., London, United Kingdom; 2Chemistry Department, Imperial College London, London, United Kingdom Trojene™ (IC-Vec Ltd/Avanti Polar Lipid Inc.) is a novel cationic liposome formulation consisting of CDAN/DOPE (50:50, m/m) Biophysical characterisation of the complexation of Trojene with pDNA revealed that Trojene/pDNA lipoplex systems are metastable Upon complexation of Trojene with pDNA, the adjustment of the unusually low pKa value of one amine group of CDAN results in structural adjustments of the lipoplex which can be monitored by circular dichroism for longer than one hour Such metastability, together with increased surface charge of Trojene lipoplexes, may contribute to more essential opsonization than observed for comparable DC-Chol/DOPE lipoplex systems This results in pronounced particle aggregation under serum conditions, which may be a beneficial element for efficient in vitro transfection A biological profile of Trojene shows low toxicity, total compatibility to carry out transfections in growth medium with no need to change medium before or after the transfection procedure and excellent transfection efficiency in a broad variety of cell lines (Keller et al, Biochemistry 2003, in press) These features altogether render this transfection reagent efficient and easy to use as a routine transfection reagent in vitro S220 MOLECULAR CONJUGATES: NEW TECHNOLOGIES 567 Efficient Delivery of Heterologous Cargos by Cationic Protein Transduction Domains Jeffrey C Mai,1 Bobby Ng,1 J Vaughn Spencer,1 Paul D Robbins.1 Department of Molecular Genetics & Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States Cationic protein transduction domains (PTDs) are short peptide sequences that are able to facilitate a rapid, receptorless internalization into nearly all cell and tissue types in vitro and in vivo Cargos delivered by PTDs range from small molecules to 200 nm liposomes Previously, PTDs were identified as naturallyoccurring motifs that mediated the translocation of the HIV-1 Tat transactivator protein and the Drosophila Antennapedia homeoprotein across cell membranes in a biologically relevant context More recent work has demonstrated that arginine homopolymers exhibit this protein transduction activity Recently, we have determined that short primary amine-containing peptides, including polylysine and polyornithine, are also able to mediate transduction of large cargos (greater than 60 kDa) into all primary cells and cell lines tested, as well as in vivo Uptake efficiencies observed for these complexes generally exceed those of L-arginine oligomers This work has reinforced the concept that cationic charge plays a key role in the internalization process We have subsequently investigated the charge-dependent entry of polyhistidine-linked cargos Histidine has a pKa = 6.04, which at physiological pH is protonated only 6% of the time However, as pH drops, the protonation state of histidine rapidly rises, reaching 61% protonation at pH 6.0 and 91% at pH 5.2 As such, we predicted that histidine would mediate cellular internalization in a pH-dependent fashion Indeed, the uptake of His6-tagged proteins, as well as polyhistidine peptide and protein complexes, demonstrates pH-sensitive cellular uptake Unlike the arginine and lysine-rich PTDs, this uptake does not occur at 4°C A hybrid peptide containing both histidine and arginine residues (8HR; HRHRHRHR) exhibits negligible uptake at pH 7.4 but demonstrates marked internalization at pH 6.0, with an efficiency that is greater than 50% that of 6R (RRRRRR), a highly active protein transduction domain 568 Evidence of Protein Transduction but Not Intercellular Transport by Proteins Fused to HIV TAT Siobhan M Cashman,1 David J Morris,1 Sonia L Sadowski,1 Rajendra Kumar-Singh.1 Ophthalmology and Human Genetics, University of Utah, Salt Lake City, UT The Human Immunodeficiency Virus (HIV-1) tat protein is known to exit virally infected cells and enter the nucleus of adjacent uninfected cells This property has been mapped to an 11 amino acid protein transduction domain (PTD) When the PTD of tat is fused to heterologous proteins and added exogenously to cells, the fusion peptide is able to demonstrate protein transduction across plasma membranes Additional studies indicate that endogenously expressed tat fusion peptides can demonstrate intercellular transport and improve biodistribution of therapeutic protein in the context of viral vectors Other studies, however, have not observed either intercellular transport or protein transduction and have been attributed to an artifact of fixation We have attempted to resolve these issues using an approach that unambiguously identifies cells that express tat fusion protein from those that receive it in their environment We tested the ability of green fluorescent protein (GFP) to be trafficked intercellularly when fused with either the full-length tat (tat-GFP) or the tat PTD (tatS-GFP) in the context of both Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy MOLECULAR CONJUGATES: NEW TECHNOLOGIES adenovirus and plasmid vectors To distinguish cells that are GFPpositive due to either infection or transfection from those that are GFP-positive due to protein transduction, we incorporated a red fluorescent protein (RFP) expression cassette in the same viral/ plasmid vector Both fluorescent microscopy and FACS analysis of live cells showed no evidence of intercellular transport either in cell culture or in vivo Both tat-GFP and tatS-GFP peptides were downregulated in their expression from adenoviral vectors by 46-57% (tat-GFP) or 14-35% (tatS-GFP), depending on cell type infected This down-regulation was not observed from plasmid vectors When the tat-GFP and tatSGFP peptides were released from infected cells and added exogenously to uninfected cells we observed protein transduction by 41% (tat-GFP) and 10% (tatS-GFP) of cells by both fluorescent microscopy of unfixed cells and FACS analysis of trypsin-treated cells This indicates a unidirectional transport of tat fusion proteins across the plasma membrane Not only are our data consistent with previous studies, they provide clarification of the seeming contradictions of previous studies 569 Novel Biodegradable PEG-PLL-g-His MultiBlock Copolymers for Non-Viral Carrier Mediated Gene Therapy Malavosklish Bikram,1 Cheol-Hee Ahn,2 Su Young Chae,3 Minhyung Lee,1 James W Yockman,1 Sung Wan Kim.1 Center for Controlled Chemical Delivery, Pharmaceutics & Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT, United States; 2School of Material Science & Engineering, Seoul National University, Seoul, Korea; 3Department of Material Science & Engineering, K-JIST, Kwangju Institute of Science & Technology, Gwangju, Korea The use of non-viral somatic gene therapy for the treatment of many severe and debilitating diseases have been very promising due to recent advances in delivery vehicle design, targeting, and endosomal escape Cationic polymers though widely used for DNA condensing ability possess certain restrictions due to the physicochemical stability of the polymer-DNA polyplexes To design a biodegradable, polymeric gene vehicle, PEG has been conjugated to the cationic poly(L-lysine) that has been synthesized via ring-opening polymerization of lysine N-carboxyanhydride Poly(ethylene glycol) (PEG) is a hydrophilic, neutral polyether whose steric stabilization properties imparts stability, improved pharmacokinetics, low toxicity and immunogenicity to molecules to which they are attached The average molecular weight (Mw) of the polymer is 41,000 with a polydispersity index of 3.19 Endosomal escape is facilitated by conjugation of N-dimethylhistidine (His) to the ε-amines of PLL at various mol% and each conjugate(5%His,9%His,16%His,22%His) was characterized by ¹H NMR The ability of the polymers to be protonated on the free ε-amines of PLL and the imidazole ring of histidines was investigated by acid-base titration that showed higher buffering capacity with increasing imidazole content in comparison to PLL Gel retardation assay confirmed the condensing ability of the conjugates at various N/P ratios; dynamic light scattering with PEG-PLL-His/pLuc complexes showed that the mean particle size ranged from 120nm to 350nm in effective diameter; surface charge of the complexes ranged from 4mV to 45mV with increasing N/P ratios; Atomic Force Microscopy (AFM) of polymer-DNA complexes on mica showed spheroidal complexes about ~100nm in length and ~100nm in width; polymer conjugates protected plasmid DNA from endonuclease degradation for at least 2hrs in vitro Protein expression via luciferase assay showed that the 16%His conjugated polymers produced four times higher protein expression in comparison to PLL in murine smooth muscle cells (A7r5) Transfection efficiency using flow cytometry with pEGFP in A7r5 showed that 10% of the cells were transfected with the 16%His conjugated polymers as opposed to 2% with PLL The cell viability Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy of A7r5 was evaluated by MTT assay and flow cytometry using propidium iodide which showed that the PEG-PLL-His treated cells had 75-100% viable cells 24 hrs after transfection as opposed to 70% viable cells for PLL treated cells CONCLUSION: Biodegradable PEG-PLL-g-His Multi-Block copolymers will be more effective gene carriers for systemic gene delivery due to the presence of PEG chains that impart increased physicochemical stability for the polymer-DNA complexes as well as the presence of the histidine moieties that are able to increase endosomal escape of the polyplexes after non-specific endocytosis This carrier can be used for systemic therapeutic gene delivery 570 Development of Novel Poly(Ethylene Glycol) Based Vehicles for Gene Therapy Anne H Schmieder,1 Leslie A Dempsey,1 Shelly E SakiyamaElbert.1 Biomedical Engineering, Washington Univeristy, St Louis, MO The aim of this research was to utilize expertise from the field of biomaterials to make synthetic vehicles for gene therapy that are less toxic and have higher transfection efficiencies than traditional cationic polymers, such as polyethylenimine (PEI) and poly-Llysine (PLL) Toward this goal, we chose poly (ethylene glycol) (PEG) as the backbone for our vehicle PEG is widely known in the biomaterials and pharmaceutical fields as a biocompatible, watersoluble polymer that can enhance the circulation time of drugs and reduce foreign body and immune responses to therapeutic agents and medical devices DNA binding peptides (DBPs) were coupled to difunctional PEG to create a PEG vehicle capable of binding DNA The goal of this study was to synthesize, characterize, and study the transfection efficiency of DBP-PEG vehicles To characterize the PEG vehicles, DBP-PEG was mixed with DNA to form particles with varying N:P charge ratios (defined as the ratio of amine groups on DBP-PEG to phosphate groups of DNA) Particle size was characterized by dynamic light scattering and found to be between 150-300nm The average zeta potential was found to be negative (-20mV) for low charge ratios (1:1-3:1) and near neutrality for N:P ratios of 3.5:1 and greater To determine the transfection capability of DBP-PEG, conditions were optimized for in vitro transfection of CHO cells β-Gal reporter gene activity was measured at 48 hr post transfection and levels of 60% that found in PEI controls (see Fig 1) were observed for optimal charge ratios (4-5:1) Since conventional cationic polymers are known to be cytotoxic, we determined the cytotoxicity of DBP-PEG/DNA particles on CHO cells and compared this with PEI and PLL controls PEG treated cells had significantly more viable cells (~95%) than either PEI (50%) or PLL(66%) controls Chloroquine was found to enhance to transfection efficiency of both PLL and the DBP-PEG vehicles in this study Although DBP-PEG transfection efficiency without chloroquine is somewhat lower than PEI, DBP-PEG is capable of achieving similar or better transfection efficiency than PLL with significantly reduced lower toxicity compared with PLL and PEI This suggests that additional functionalities that enhance the ability of our DBPPEG vehicles to promote endosomal escape could improve their transfection efficiency The PEG-based vehicles proposed above, provide a potentially non-toxic alternative to the currently available non-viral gene therapy agents with similar transfection efficiency to currently available cationic polymers Characteristics of polymers used for in-vitro transfection Mol Wt charge Avg Zeta Avg.% ratio size potential cell (N+/P-) (nm) (mV) viability PEI 50,000 1.2:1 261.3 +15.3 50.2 +/-23.9 PLL 30,300 2:1 205.1 +22 66.2 +/-27.3 DBP-PEG 8704 4:1 230.1 +0.37 95.8 +/-1.8 DBP-PEG+Chl 8704 4:1 208.1 +0.37 n/a Relative β-Gal activity 1.0 0.13 0.57 3.4 S221 ... added exogenously to uninfected cells we observed protein transduction by 41% (tat- GFP) and 10% (tatS-GFP) of cells by both fluorescent microscopy of unfixed cells and FACS analysis of trypsin-treated... from adenoviral vectors by 46-57% (tat- GFP) or 14-35% (tatS-GFP), depending on cell type infected This down-regulation was not observed from plasmid vectors When the tat- GFP and tatSGFP peptides... viral/ plasmid vector Both fluorescent microscopy and FACS analysis of live cells showed no evidence of intercellular transport either in cell culture or in vivo Both tat- GFP and tatS-GFP peptides