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• W2075584549 abstract "Chimeric RNA/DNA oligonucleotides have been shown to promote single nucleotide exchange in genomic DNA. A chimeric molecule was designed to introduce an A to C nucleotide conversion at the Ser365 position of the rat factor IX gene. The oligonucleotides were encapsulated in positive, neutral, and negatively charged liposomes containing galactocerebroside or complexed with lactosylated polyethyleneimine. The formulations were evaluated for stability and efficiency in targeting hepatocytes via the asialoglycoprotein receptor. Physical characterization and electron microscopy revealed that the oligonucleotides were efficiently encapsulated within the liposomes, with the positive and negative formulations remaining stable for at least 1 month. Transfection efficiencies in isolated rat hepatocytes approached 100% with each of the formulations. However, the negative liposomes and 25-kDa lactosylated polyethyleneimine provided the most intense nuclear fluorescence with the fluorescein-labeled oligonucleotides. The lactosylated polyethyleneimine and the three different liposomal formulations resulted in A to C conversion efficiencies of 19–24%. In addition, lactosylated polyethyleneimine was also highly effective in transfecting plasmid DNA into isolated hepatocytes. The results suggest that both the liposomal and polyethyleneimine formulations are simple to prepare and stable and give reliable, reproducible results. They provide efficient delivery systems to hepatocytes for the introduction or repair of genetic mutations by the chimeric RNA/DNA oligonucleotides. Chimeric RNA/DNA oligonucleotides have been shown to promote single nucleotide exchange in genomic DNA. A chimeric molecule was designed to introduce an A to C nucleotide conversion at the Ser365 position of the rat factor IX gene. The oligonucleotides were encapsulated in positive, neutral, and negatively charged liposomes containing galactocerebroside or complexed with lactosylated polyethyleneimine. The formulations were evaluated for stability and efficiency in targeting hepatocytes via the asialoglycoprotein receptor. Physical characterization and electron microscopy revealed that the oligonucleotides were efficiently encapsulated within the liposomes, with the positive and negative formulations remaining stable for at least 1 month. Transfection efficiencies in isolated rat hepatocytes approached 100% with each of the formulations. However, the negative liposomes and 25-kDa lactosylated polyethyleneimine provided the most intense nuclear fluorescence with the fluorescein-labeled oligonucleotides. The lactosylated polyethyleneimine and the three different liposomal formulations resulted in A to C conversion efficiencies of 19–24%. In addition, lactosylated polyethyleneimine was also highly effective in transfecting plasmid DNA into isolated hepatocytes. The results suggest that both the liposomal and polyethyleneimine formulations are simple to prepare and stable and give reliable, reproducible results. They provide efficient delivery systems to hepatocytes for the introduction or repair of genetic mutations by the chimeric RNA/DNA oligonucleotides. oligonucleotide asialoglycoprotein receptor gas phase electrophoretic mobility molecular analysis polyethyleneimine lactosylated polyethyleneimine Recently, a novel technology for facilitating targeted gene correction of episomal DNA in mammalian cells has been described (1Kmiec E.B. Adv. Drug Delivery Reviews. 1995; 17: 333-340Crossref Scopus (3) Google Scholar,2Yoon K. Cole-Strauss A. Kmiec E.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2071-2076Crossref PubMed Scopus (215) Google Scholar). The technology involves the use of a chimeric oligonucleotide (ON)1 composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with poly(T) hairpin caps at both ends and a 3′ G:C clamp. In a typical heteroduplex molecule, two blocks of 10 2′-O-methyl RNA residues flank a pentameric stretch of DNA. The structure exhibits increased chemical and thermal stability as well as greater resistance to a variety of nucleases. The chimeric molecule is designed so that it aligns in perfect register with a specified genome target, with the exception of a single base pair in the region of the pentameric stretch of DNA. The single mismatched nucleotide is recognized by endogenous DNA repair systems, thus affecting alteration of the DNA sequence of the targeted gene (1Kmiec E.B. Adv. Drug Delivery Reviews. 1995; 17: 333-340Crossref Scopus (3) Google Scholar, 3Cole-Strauss A. Nöe A. Kmiec E.B. Antisense Nucleic Acid Drug Dev. 1997; 7: 211-216Crossref PubMed Scopus (11) Google Scholar). This technology has been shown to be successful in vitro in correcting the mutation responsible for sickle cell anemia (4Cole-Strauss A. Yoon K. Xiang Y. Byrne B.C Rice M.C. Gryn J. Holloman W.K. Kmiec E.B. Science. 1996; 273: 1386-1389Crossref PubMed Scopus (282) Google Scholar) and in mutating the rat factor IX gene in rat hepatocytes in vivo (5Kren B.T. Bandyopadhyay P. Steer C.J. Nat. Med. 1998; 4: 285-290Crossref PubMed Scopus (245) Google Scholar). While the efficiency of nucleotide conversion may depend on a number of factors, delivery of DNA to the cell and its nucleus remains key. In designing an optimal delivery system, important parameters include cell toxicity and targeting specificity as well as stability of the formulation. The delivery of intact nucleic acids from the extracellular environment to the nucleus has remained a major barrier to long term and stable gene expression both ex vivo and in vivo. After cellular uptake of nucleic acids by endocytosis, only a small fraction of the molecules reach the nucleus intact (6Loke S.L. Stein C.A. Zhang X.H. Mori K. Nakanishi M. Subasinghe C. Cohen J.S. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3474-3478Crossref PubMed Scopus (736) Google Scholar, 7Chin D.J. Green G.A. Zon G. Szoka Jr., F.C. Straubinger R.M. New Biol. 1990; 2: 1091-1100PubMed Google Scholar). Interestingly, a variety of studies, including direct cytoplasmic injection of nucleic acids, suggest that these molecules display significant nuclear tropism (7Chin D.J. Green G.A. Zon G. Szoka Jr., F.C. Straubinger R.M. New Biol. 1990; 2: 1091-1100PubMed Google Scholar, 8Leonetti J.P. Mechti N. Degols G. Gagnor C. Lebleu B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2702-2706Crossref PubMed Scopus (349) Google Scholar, 9Fisher T.L. Terhorst T. Cao X. Wagner R.W. Nucleic Acids Res. 1993; 21: 3857-3865Crossref PubMed Scopus (231) Google Scholar). However, the movement of DNA from the cytoplasm to the nucleus continues to be an important limitation to successful gene transfer (10Zabner J. Fasbender A.J. Moninger T. Poellinger K.A. Welsh M.J. J. Biol. Chem. 1995; 270: 18997-19007Abstract Full Text Full Text PDF PubMed Scopus (1309) Google Scholar). Cationic lipids and polycations have been used to complex nucleic acids, thereby protecting them from degradation, while simultaneously increasing their endocytic uptake into cells (11Felgner P.L. Gadek T.R. Holm M. Roman R. Chan H.W. Wenz M. Northrop J.P. Ringold G.M. Danielsen M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7413-7417Crossref PubMed Scopus (4388) Google Scholar, 12Bennett C.F. Chiang M.-Y. Chan H. Shoemaker J.E.E. Mirabelli C.K. Mol. Pharmacol. 1992; 41: 1023-1033PubMed Google Scholar, 13Capaccioli S. Di Pasquale G. Mini E. Mazzei T. Quattrone A. Biochem. Biophys. Res. Commun. 1993; 197: 818-825Crossref PubMed Scopus (161) Google Scholar, 14Felgner J.H. Kumar R. Sridhar C.N. Wheeler C.J. Tsai Y.J. Border R. Ramsey P. Martin M. Felgner P.L. J. Biol. Chem. 1994; 269: 2550-2561Abstract Full Text PDF PubMed Google Scholar). Many of these lipid formulations, although successful in cell culture, have not been useful for in vivo delivery because of short serum half-life, toxicity, and lack of tissue specificity (15Gao X. Huang L. Gene Ther. 1995; 2: 710-722PubMed Google Scholar). Polycations, such as poly-l-lysine, polyethyleneimine (PEI), and polyamino lipids, form water-soluble complexes that can provide simple but very efficient delivery systems. The presence of free amino groups on these agents makes them amenable to chemical modification for the attachment of ligands capable of targeting specific tissues. For example, asia- loorosomucoid (16Findeis M.A. Wu C.H. Wu G.Y. Methods Enzymol. 1994; 247: 341-351Crossref PubMed Scopus (29) Google Scholar, 17Martinez-Fong D. Mullersman J.E. Purchio A.F. Armendariz-Borunda J. Martinez-Hernandez A. Hepatology. 1994; 20: 1602-1608Crossref PubMed Scopus (57) Google Scholar) and galactose (18Perales J.C. Grossmann G.A. Molas M. Liu G. Ferkol T. Harpst J. Oda H. Hanson R.W. J. Biol. Chem. 1997; 272: 7398-7407Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) have been conjugated to poly-l-lysine for targeting to the asialoglycoprotein receptor (ASGPR) on hepatocytes. The asialoorosomucoid-poly-l-lysine system has been demonstrated to increase hepatocyte-directed gene delivery bothin vitro (19Wu G.Y. Wu C.H. Biochemistry. 1988; 27: 887-892Crossref PubMed Scopus (217) Google Scholar) and in vivo (20Chowdhury N.R. Wu C.H. Wu G.Y. Yerneni P.C. Bommineni V.R. Chowdhury J.R. J. Biol. Chem. 1993; 268: 11265-11271Abstract Full Text PDF PubMed Google Scholar). A similar strategy has been utilized for hepatocyte-specific gene delivery using lipopolyamine-condensed DNA targeted with galactose ligands (21Remy J.-S. Kichler A. Mordvinov V. Schuber F. Behr J.-P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1744-1748Crossref PubMed Scopus (276) Google Scholar). Transfection of different cell types with the chimeric ONs has been accomplished with dioleoyl trimethylammoniumpropane (4Cole-Strauss A. Yoon K. Xiang Y. Byrne B.C Rice M.C. Gryn J. Holloman W.K. Kmiec E.B. Science. 1996; 273: 1386-1389Crossref PubMed Scopus (282) Google Scholar), Lipofectin® (2Yoon K. Cole-Strauss A. Kmiec E.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2071-2076Crossref PubMed Scopus (215) Google Scholar), and PEI (22Kren B.T. Cole-Strauss A. Kmiec E.B. Steer C.J. Hepatology. 1997; 25: 1462-1468Crossref PubMed Scopus (125) Google Scholar). However, these systems lack the physicochemical stability and proper formulation for efficientin vivo delivery. The existing system of gene delivery by cationic lipids consists primarily of heterogeneous complexes and aggregates rather than liposome-encapsulated material (23Smith J.G. Walzem R.L. German J.B. Biochim. Biophys. Acta. 1993; 1154: 327-340Crossref PubMed Scopus (138) Google Scholar). A combinatorial approach of condensing the DNA using polycationic molecules followed by liposome association or encapsulation has been demonstrated to greatly increase the efficacy of the delivery systems (24Zhou X. Huang L. Biochim. Biophys. Acta. 1994; 1189: 195-203Crossref PubMed Scopus (493) Google Scholar, 25Gao X. Huang L. Biochemistry. 1996; 35: 1027-1036Crossref PubMed Scopus (452) Google Scholar, 26Lee R.J. Huang L. J. Biol. Chem. 1996; 271: 8481-8487Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). In the present study, we describe the preparation and characterization of three liposomal systems, targeted to the ASGPR on rat hepatocytes with galactocerebroside, for delivery of the chimeric molecules. In addition, we have characterized a nonliposomal targeting system using lactosylated PEI. In fact, unmodified PEI has been shown to be a useful gene delivery vehicle bothin vitro and in vivo (27Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.-P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5654) Google Scholar, 28Abdallah B. Hassan A. Benoist C. Goula D. Behr J.P. Demeneix B.A. Human Gene Ther. 1996; 7: 1947-1954Crossref PubMed Scopus (529) Google Scholar, 29Boletta A. Benigni A. Lutz J. Remuzzi G. Soria M.R. Monaco L. Human Gene Ther. 1997; 8: 1243-1251Crossref PubMed Scopus (186) Google Scholar). Our results indicate that RNA/DNA ONs can be efficiently delivered to hepatocytes for gene conversion by both liposomal and nonliposomal systems without the use of viral vectors. Chimeric RNA/DNA factor IX ONs were synthesized by Applied Biosystems, Inc. (Foster City, CA) as described previously (2Yoon K. Cole-Strauss A. Kmiec E.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2071-2076Crossref PubMed Scopus (215) Google Scholar). The crude ONs were purified by high pressure liquid chromatography and quantitated by UV absorbance. Greater than 95% of the purified ONs were determined to be full-length. A 68-mer all-DNA chimeric ON was obtained from Genosys Biotechnologies, Inc. (The Woodlands, TX). The molecules were 3′-end-labeled using terminal transferase and fluorescein-12-dUTP from Boehringer Mannheim according to the manufacturer's recommendation. Labeled ONs were then mixed with unlabeled ONs at a 2:3 ratio. Dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dioleoyl phosphatidylserine, galactocerebroside, and dioleoyl trimethylammoniumpropane were purchased from Avanti Polar Lipids Inc. (Alabaster, AL) as dried powders. Stock solutions (2 mg/ml) of the lipids and lipid films were prepared as described previously (30Bandyopadhyay P. Kren B.T. Ma X. Steer C.J. BioTechniques. 1998; 25: 282-292Crossref PubMed Scopus (41) Google Scholar). Positively charged liposomes consisted of dioleoyl phosphatidylcholine/dioleoyl trimethylammoniumpropane/galactocerebroside at a 6:1:0.56 molar ratio; neutral liposomes included dioleoyl phosphatidylethanolamine/dioleoyl phosphatidylcholine/galactocerebroside at a 1:1:0.16 ratio, and negatively charged liposomes were formulated with dioleoyl phosphatidylserine/dioleoyl phosphatidylcholine/galactocerebroside at a molar ratio of 1:1:0.16. PEI (800 kDa) was purchased from Fluka Chemical Corp. (Ronkonkoma, NY). A stock solution of 0.05 mPEI monomer corresponding to ∼50 nmol of PEI amine/μl (27Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.-P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5654) Google Scholar) was prepared in Milli Q® (Millipore Corp., Bedford, MA) water, and its pH was adjusted to 7.6 with 6 m HCl. PEI (4 nmol of amine/nmol of RNA/DNA phosphate) and either unlabeled or fluorescently labeled chimeric molecules (100–250 μg) were each diluted into 250 μl of 0.15 m NaCl. The solutions were vortexed separately for 5–10 min at room temperature followed by the dropwise addition of the PEI solution to the ON, with continual vortexing for an additional 5–10 min. The PEI complex was then added to the positively charged lipid film. For the neutral and negatively charged liposomes, naked or fluorescently labeled chimeric ONs (150 μg) were diluted into 500 μl of 0.15 m NaCl and added to the lipid film. The films were hydrated by alternate vortex mixing and warming until a homogeneous suspension was formed. The resulting suspension was extruded through a series of polycarbonate membranes down to a pore size of 0.05 μm using a Liposofast® miniextruder system (Avestin, Inc., Ottawa, Canada). The size of the extruded liposomes was determined by light scattering using a NICOMP 370 submicron particle size analyzer (Pacific Scientific Instruments, Santa Barbara, CA). In short, the Brownian motion of suspended particles modulates the phase of scattered light waves from a laser beam. Thus, the intensity of the detected light fluctuates in a random fashion and is detected with a photomultiplier tube. For a given particle, the average lifetime for the intensity fluctuations is roughly equal to the average time required for two particles to change their separation by one-half of the fixed laser wavelength λ. The spherical radius of the particle is then obtained from the diffusion coefficient of the particle over time using the Stokes-Einstein relation. The phospholipid concentrations in the liposomal preparations were determined by the method of Stewart (31Stewart J.C.M. Anal. Biochem. 1980; 104: 10-14Crossref PubMed Scopus (1544) Google Scholar). Aliquots of 20–50 μl were diluted to 500 μl using Milli Q® water, and the phospholipids were extracted twice using 500 μl of chloroform:methanol (1:1 v/v). The liposomal extracts were also analyzed for PEI complexes and naked ONs following ultrafiltration to remove the nonencapsulated material. Briefly, 40–50 μl of liposome suspension after lipid extraction was diluted with 300 μl of heparin (1 unit/μl) and incubated at 37 °C for 6 h. Following phenol/chloroform extraction, the aqueous phase was precipitated at −20 °C overnight following the addition of 80 μl of 3 m sodium acetate (pH 5.2), 500 μl isopropyl alcohol, 10 μg of tRNA, and 300 μl of RNAmate® (Intermountain Scientific, Inc., Kaysville, UT). An aliquot of the stock PEI·ON complex was subjected to the same extraction procedure. The ONs were analyzed by 4% low melting point agarose gel electrophoresis containing 1 μg/ml ethidium bromide and visualized using UV light. The relative intensities of the bands were analyzed by densitometry using a Bio-Rad model GS-700 imaging densitometer. PEI was lactosylated by a modification of a previously described method for oligosaccharide conjugation (32Gray G.R. Arch. Biochem. Biophys. 1974; 163: 426-428Crossref PubMed Scopus (224) Google Scholar). Briefly, 3–5 ml of a 0.1–0.2 m stock of either 800- or 25-kDa PEI (Aldrich) in 0.2 m ammonium acetate or Tris-HCl, pH 7.6, buffer solution was incubated with 7–8 mg of sodium cyanoborohydride (Sigma) and approximately 20–30 mg of lactose monohydrate (Sigma) at 37 °C for 10 days. The reaction mixture was dialyzed against Milli Q® water for 48 h with 1–2 changes of water/day. The amount of sugar (as galactose) conjugated with PEI was determined by the phenol-sulfuric acid method (33Dubois M. Gilles K.A. Hamilton J.K. Rebers P.A. Smith F. Anal. Chem. 1956; 28: 350-356Crossref Scopus (41197) Google Scholar). The number of moles of free secondary amines in the lactosylated PEI (L-PEI) was determined as follows. A standard curve was generated using a 0.02 m stock solution of PEI; several aliquots of the stock were diluted to 1 ml using Milli Q® water in glass tubes, and then 50 μl of ninhydrin reagent (Sigma) was added to each tube and vortexed vigorously for 10 s. Color development was allowed to proceed in the dark at room temperature for 10–12 min, and the OD was determined at 485 nm on a Beckman DU-64 spectrophotometer. An equivalent of 5 nmol of amine as L-PEI and 5 nmol as PEI per nmol of RNA/DNA phosphate were mixed, diluted in 0.15 m NaCl as required, and used for transfection as described previously (5Kren B.T. Bandyopadhyay P. Steer C.J. Nat. Med. 1998; 4: 285-290Crossref PubMed Scopus (245) Google Scholar). For the in vitrofluorescently labeled ON uptake experiments, the 25-kDa PEI plus L-PEI mix (1:1 molar ratio of amines) was complexed with the labeled chimeric molecules at a ratio of 10 or 6 nmol of PEI amines per nmol of DNA/RNA phosphate in 5% dextrose. For the in vivo studies, the 25-kDa PEI plus L-PEI mix (1:1 molar ratio of amines) was complexed with the RNA/DNA ONs at the 6:1 amine:phosphate ratio in 5% dextrose. Nuclease resistance of the complexed RNA/DNA molecules was determined by treating the samples with 40 units of RNase A and 40 units of RQ1 RNase free DNase (Promega Corp., Madison, WI) for 40 min at 37 °C. The reaction was terminated using 2 μl each of 0.5 m EGTA and 0.5 m EDTA. Following two phenol/chloroform extractions, the complexes were dissociated using heparin (50 units/μg of nucleic acid) at 37 °C for 60 min, and the products were analyzed on a 4% low melting point agarose gel. The size of the chimeric molecules alone or complexed to PEI was determined by gas phase electrophoretic mobility molecular analysis (GEMMA) (TSI Inc., Minneapolis, MN) (34Kaufman S.L. Skogen J.W. Dorman F.D. Zarrin F. Anal. Chem. 1996; 68: 1895-1904Crossref PubMed Scopus (189) Google Scholar) or by light scattering measurements. For GEMMA, chimeric ONs (4 mg/ml) were diluted at a 1:3,000 ratio with 0.02m ammonium acetate buffer. The diluted suspension was then transformed into an aerosol by electrospray drying. The resulting high charge on the molecules was neutralized by a radioactive α-emitter, and the singly charged chimeric molecules were size-separated according to their mobility in air. Liposome suspensions were applied as a drop on glow-discharged formvar carbon-coated grids (300 or 400 mesh, Polysciences Inc., Warrington, PA) and negatively stained using 2% ammonium molybdate. PEI complexes were stained with either a 1% uranyl acetate or a 2% ammonium molybdate solution, and the samples were visualized using a JEOL100-CX electron microscope. For scanning electron microscopy, samples were mounted in drops onto borosilicate chips and fixed with 2% osmium tetroxide vapors for 2 h at 25 °C, followed by stepwise dehydration in ethanol (35Hamilton-Attwell V.L. Du Plessis J. van Wyk C.J. J. Microscopy. 1987; 145: 347-349PubMed Google Scholar). The mounted samples were stored in 100% ethanol until critical point drying in liquid carbon dioxide. Immediately following drying, samples were sputter-coated with platinum metal for 6 min at 4.2 V. Samples were viewed the following day on a Hitachi S-900 field emission scanning electron microscope at 1.5 kV at magnifications of × 1,200–200,000. For freeze-fracture analysis, anionic liposomes were made using fluorescein-, Cy3-labeled, or naked ONs, frozen in freon, and then stored in liquid nitrogen until fracture. Fracture was performed on a BAF 060 apparatus. The fractured surfaces were coated with 6 nm of platinum at a 45° angle to the surface, followed by 20 nm of carbon at a 90° angle to the surface. Replicas were cleaned overnight by flotation in 1 m sodium hypochlorite and then rinsed for 1 h in water. After transfer to 200 mesh carbon grids, the specimens were viewed at 80 kV on a JE0L100-CX electron microscope. Primary rat hepatocytes were isolated from male Sprague-Dawley rats (125–175 g) (Harlan Sprague-Dawley, Inc., Indianapolis, IN) using a two-step collagenase perfusion procedure described previously (36Mariash C.N. Seelig S. Schwartz H.L. Oppenheimer J.H. J. Biol. Chem. 1986; 261: 9583-9586Abstract Full Text PDF PubMed Google Scholar). The hepatocytes were plated at a density of 4 × 105 cells/35 × 10-mm PrimariaTMdish (Becton-Dickinson Labware, Lincoln Park, NJ) and maintained for 18–24 h prior to transfection in William's E medium (Life Technologies, Inc.) supplemented with l-glutamine, 0.01 units/ml insulin, 2 mm Hepes, 23 mmNaHCO3, 0.01 μm dexamethasone, and 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Norcross, GA). Positive, neutral, and negative liposomes were diluted with Hepes-buffered saline, pH 7.4, to a final concentration of 2–6 μg of ON/100 μl and used for transfection as described previously (30Bandyopadhyay P. Kren B.T. Ma X. Steer C.J. BioTechniques. 1998; 25: 282-292Crossref PubMed Scopus (41) Google Scholar). Nonencapsulated PEI·ON complexes at identical amine:phosphate ratios were used as positive controls and diluted as described above. The cells were washed twice with William's E medium further supplemented with an additional 2.5 mmCaCl2 and then incubated with 1 ml of the same medium. An aliquot of 100 μl of transfecting solution was then added to each 35-mm dish. After 18 h of transfection, an additional 2 ml of medium containing 10% heat-inactivated FBS was added, and the cells were maintained for an additional 6–30 h. The reporter plasmid PGL3 (Promega Corp.) encoding the luciferase gene was prepared in 0.15m NaCl or 5% dextrose using various ratios ofL-PEI to 25-kDa PEI amines as well as nucleic acid phosphate to PEI amines. Hepatocytes were harvested 48 h after transfection using 200 μl of reporter lysis buffer, and the luciferase activity was determined by the luciferase assay reagent (Promega) according to the manufacturer's protocol. Protein was quantitated with the Bio-Rad protein assay reagent as specified by the manufacturer. Hepatocytes transfected with the fluorescently labeled ONs were fixed 24 h post-transfection in phosphate-buffered saline, pH 7.4, containing 4% paraformaldehyde (w/v) for 10 min at room temperature. Following fixation, the cells were counterstained using DiI (Molecular Probes, Inc., Eugene, OR) and coverslipped using SlowFade™ (Molecular Probes) antifade mounting medium in phosphate-buffered saline and examined using a MRC1000 confocal microscope (Bio-Rad) (22Kren B.T. Cole-Strauss A. Kmiec E.B. Steer C.J. Hepatology. 1997; 25: 1462-1468Crossref PubMed Scopus (125) Google Scholar). Male Sprague-Dawley rats (Harlan Sprague-Dawley) (∼65 g) were maintained on a standard 12-h light-dark cycle and fed ad libitum standard laboratory chow. The rats were restrained by hand, and the chimeric molecules, complexed with 25-kDa PEI/L-PEI (1:1 molar ratio of amines) at a ratio of 6 equivalents of PEI nitrogen per RNA/DNA phosphate in 500 μl of 5% dextrose (28Abdallah B. Hassan A. Benoist C. Goula D. Behr J.P. Demeneix B.A. Human Gene Ther. 1996; 7: 1947-1954Crossref PubMed Scopus (529) Google Scholar) were administered in vivo by tail vein injection. Vehicle controls received an equal volume of PEI in Tris-HCl, pH 7.6. The animals were anesthesized with ether 1 and 18 weeks postinjection and underwent a midline incision. The liver was exposed, and random samples were excised for DNA isolation. After 3 weeks, two of the animals received an additional injection of 500 μg of the L-PEI/chimeric complexes followed 24 h later by 70% partial hepatectomy (37Higgins G.M. Anderson R.M. Arch. Pathol. 1931; 12: 186-202Google Scholar) to determine the replicative stability of the genomic nucleotide conversion. Genomic DNA larger than 100–150 base pairs was isolated from the tissue samples using the high pure PCR template preparation kit (Boehringer Mannheim) for PCR amplification of exon 8 of the rat factor IX gene. Blood samples were obtained from the test groups at varying times up to 52 weeks after the final tail vein injection and mixed in 0.1 volume of 0.105 m sodium citrate/citric acid. The blood samples were centrifuged at 2,500 × g, followed by 15,000 × gcentrifugation, and the resulting plasma was stored at −70 °C. The factor IX activity was determined from activated partial thromboplastin time assays as described previously (5Kren B.T. Bandyopadhyay P. Steer C.J. Nat. Med. 1998; 4: 285-290Crossref PubMed Scopus (245) Google Scholar). The factor IX activity of duplicate samples was determined from a log-log standard curve constructed from the activated partial thromboplastin time results of pooled plasma from 12 similarly aged normal male rats through multiple dilutions. The chimeric ON- and vehicle-transfected cells were harvested by scraping 48 h after transfection. Genomic DNA larger than 100–150 base pairs was isolated using the high pure PCR template preparation kit (Boehringer Mannheim). The isolated DNA (500 ng) from either primary hepatocytes or liver was used for PCR amplification of a 374-nucleotide fragment of the rat factor IX gene as described previously (5Kren B.T. Bandyopadhyay P. Steer C.J. Nat. Med. 1998; 4: 285-290Crossref PubMed Scopus (245) Google Scholar). The primers were designed as 5′-ATTGCCTTGCTGGAACTGGATAAAC-3′ and 5′-TTGCCTTTCATTGCACATTCTTCAC-3′ (Oligos Etc., Wilsonville, OR) corresponding to nucleotides 433–457 and 782–806, respectively, of the rat factor IX cDNA (38Sarkar G. Koeberl D.D. Sommer S.S. Genomics. 1990; 6: 133-143Crossref PubMed Scopus (49) Google Scholar). The PCR amplicons were subcloned into the TA cloning vector pCR™2.1 (Invitrogen, San Diego, CA), and the ligated material was used to transform frozen competent E. coli. 18–20 h after plating, the colonies were lifted onto MSI MagnaGraph nylon filters, replicated, and processed for hybridization according to the manufacturer's recommendation. The filters were hybridized with 17-mer ON probes 365A (5′-AAGGAGATAGTGGGGGA-3′) or 365C (5′-AAGGAGATCGTGGGGGA-3′) (Life Technologies), where the targeted nucleotide for mutagenesis is underlined. The probes were 32P-end-labeled using [γ-32P]ATP (>7,000 Ci/mmol) and T4 polynucleotide kinase (New England Biolabs, Inc., Beverly, MA). Hybridizations were performed at 37 °C, and the filters were processed posthybridization as described previously (5Kren B.T. Bandyopadhyay P. Steer C.J. Nat. Med. 1998; 4: 285-290Crossref PubMed Scopus (245) Google Scholar, 39Melchoir Jr., W.B. Von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 298-302Crossref PubMed Scopus (173) Google Scholar). Autoradiography was performed with NEN® Reflection film at −70 °C using an intensifying screen. Plasmid DNA was prepared from colonies hybridizing to either 365A or 365Cusing a Qiagen miniprep kit (Chatsworth, CA). Sequencing was performed with an ABI 370A sequencer (Perkin-Elmer, Corp., Foster City, CA) using a gene-specific primer, 5′-GTTGACCGAGCCACATGCCTTAG-3′, corresponding to nucleotides 616–638 of the rat factor IX cDNA (38Sarkar G. Koeberl D.D. Sommer S.S. Genomics. 1990; 6: 133-143Crossref PubMed Scopus (49) Google Scholar), and the mp13 forward and reverse primers. Data were analyzed using InStat version 2.01 (GraphPad Software, San Diego, CA) to calculate analysis of variance, and probability (p) values were determined using Bonferroni multiple comparisons. Two column comparisons were analyzed using Welch's alternate t test. By GEMMA the chimeric ONs ranged in size from 4 to 7 nm, with major peaks at 4.6 and 5.8 nm (Fig.1 a). After heating to 95 °C and then cooling to room temperature, the chimeric molecules exhibited a unimodal distribution with an average diameter of 4.4 nm (Fig.1 b). ONs complexed with PEI in 0.15 m NaCl ranged in size from 20 to 200 nm. Electron microscopy revealed that the complexes were often aggregated into large c" @default.
• W2075584549 created "2016-06-24" @default.
• W2075584549 creator A5007378745 @default.
• W2075584549 creator A5042616886 @default.
• W2075584549 creator A5069186777 @default.
• W2075584549 creator A5071553397 @default.
• W2075584549 creator A5089972728 @default.
• W2075584549 date "1999-04-01" @default.
• W2075584549 modified "2023-10-17" @default.
• W2075584549 title "Nucleotide Exchange in Genomic DNA of Rat Hepatocytes Using RNA/DNA Oligonucleotides" @default.
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