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INTRODUCTION |
Recently, a novel technology for facilitating targeted gene
correction of episomal DNA in mammalian cells has been described (1,
2). 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 (1, 3). This technology has been shown to be
successful in vitro in correcting the mutation responsible
for sickle cell anemia (4) and in mutating the rat factor IX gene in
rat hepatocytes in vivo (5). 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 (6, 7). Interestingly, a
variety of studies, including direct cytoplasmic injection of nucleic
acids, suggest that these molecules display significant nuclear tropism
(7-9). However, the movement of DNA from the cytoplasm to the nucleus
continues to be an important limitation to successful gene transfer
(10). Cationic lipids and polycations have been used to complex nucleic
acids, thereby protecting them from degradation, while simultaneously
increasing their endocytic uptake into cells (11-14). 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 (15). 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 (16, 17) and galactose (18) 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 both
in vitro (19) and in vivo (20). A similar
strategy has been utilized for hepatocyte-specific gene delivery using lipopolyamine-condensed DNA targeted with galactose ligands (21).
Transfection of different cell types with the chimeric ONs has been
accomplished with dioleoyl trimethylammoniumpropane (4), Lipofectin® (2), and PEI (22). However, these systems lack
the physicochemical stability and proper formulation for efficient
in vivo delivery. The existing system of gene delivery by
cationic lipids consists primarily of heterogeneous complexes and
aggregates rather than liposome-encapsulated material (23). 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
(24-26). 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 both
in vitro and in vivo (27-29). 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.
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EXPERIMENTAL PROCEDURES |
Synthesis and Fluorescent Labeling of RNA/DNA ONs--
Chimeric
RNA/DNA factor IX ONs were synthesized by Applied Biosystems, Inc.
(Foster City, CA) as described previously (2). 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.
Liposomal Formulations--
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 (30). 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 M
PEI monomer corresponding to ~50 nmol of PEI amine/µl (27) 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).
Analysis of the Liposome Formulations--
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
. 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 (31). 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.
Preparation of Lactosylated PEI--
PEI was lactosylated by a
modification of a previously described method for oligosaccharide
conjugation (32). 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
(33). 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.
Preparation of L-PEI·ON Complexes--
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 (5). For the in vitro
fluorescently 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.
Size Analysis of the ON and L-PEI·ON Complexes--
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) (34) or by light scattering measurements. For
GEMMA, chimeric ONs (4 mg/ml) were diluted at a 1:3,000 ratio with 0.02 M 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
-emitter,
and the singly charged chimeric molecules were size-separated according
to their mobility in air.
Electron Microscopy--
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 (35). 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.
Cell Cultures--
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 (36). The hepatocytes were plated at a density of 4 × 105 cells/35 × 10-mm PrimariaTM
dish (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 mM
NaHCO3, 0.01 µM dexamethasone, and 10%
heat-inactivated fetal bovine serum (Atlanta Biologicals, Norcross, GA).
Transfections--
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 (30). 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 mM
CaCl2 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.
Transfection of Plasmids with L-PEI--
The reporter plasmid
PGL3 (Promega Corp.) encoding the luciferase gene was prepared in 0.15 M NaCl or 5% dextrose using various ratios of
L-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.
Nuclear Uptake of Fluorescently Labeled ONs--
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 SlowFadeTM (Molecular Probes)
antifade mounting medium in phosphate-buffered saline and examined
using a MRC1000 confocal microscope (Bio-Rad) (22).
In Vivo Delivery of L-PEI Complexes--
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 (28) 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 (37) 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.
Factor IX Activity--
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 × g centrifugation, and the resulting plasma was stored at
70 °C. The
factor IX activity was determined from activated partial thromboplastin time assays as described previously (5). 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.
DNA Isolation and Cloning--
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 (5). 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 (38). The PCR
amplicons were subcloned into the TA cloning vector pCRTM2.1
(Invitrogen, San Diego, CA), and the ligated material was used to
transform frozen competent E. coli.
Colony Lift Hybridization and Sequencing--
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 [
-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 (5, 39).
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 365C
using 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 (38), and the mp13
forward and reverse primers.
Statistical Analysis--
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.
 |
RESULTS |
Characterization of the Liposomal Formulations and PEI/L-PEI
Complexes--
By GEMMA the chimeric ONs ranged in size from 4 to 7 nm, with major peaks at 4.6 and 5.8 nm (Fig.
1a). 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.
1b). 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 clusters (Fig. 2,
a and c), thus
explaining the wide range in their size distribution. However, when the
complexes were prepared in 5% dextrose, they appeared as smaller
discrete particles with a mean diameter of ~20 nm (Fig. 2,
b and d). Following size-selected extrusion, the liposomes showed a unimodal size distribution with a volume-weighted mean diameter of <50 nm by light scattering. Neither the anionic nor
cationic liposomes changed in size after overnight incubation at
37 °C or storage for 1 month at 4 °C. In contrast, the neutral liposomes aggregated and fused into larger vesicles under these conditions. The uniformity of anionic and cationic liposome suspensions was confirmed by electron microscopy. The PEI·ON complexes were either situated within the bilayer of the cationic liposomes or more
typically entirely encapsulated within the liposome core (Fig.
3a). Negative liposomes
prepared with the naked chimeric molecules appeared as uniform vesicles
with small electron dense particles randomly distributed within the
vesicles (Fig. 3b). The encapsulation of ONs was confirmed
by freeze-fracture analysis of the anionic liposomes (Fig. 3,
c and d).

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Fig. 1.
Size analysis of chimeric RNA/DNA
molecules. Lyophilized RNA/DNA ONs were solubilized in water at a
concentration of 4 mg/ml and stored at 80 °C. After slowly thawing
on ice, the molecules were heated to 95 °C for 10 min and then
cooled to room temperature. A 1:3,000 dilution of the ONs in 0.02 M ammonium acetate buffer was processed for GEMMA prior to
(a) and after heating (b). The unheated sample
exhibited two distinct peaks of 4.6 and 5.8 nm, whereas the heated
sample consisted of a single peak of 4.4 nm.
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Fig. 2.
Transmission electron microscopy and scanning
electron microscopy of PEI·ON complexes. a,
L-PEI·ON complexes in 0.15 M NaCl were
applied to glow-discharged formvar carbon-coated grids and stained with
1% uranyl acetate and visualized by transmission electron microscopy
as described under "Experimental Procedures." c,
L-PEI·ON complexes in 0.15 M NaCl were
visualized by scanning electron microscopy as described under
"Experimental Procedures." b and d,
L-PEI·ON complexes in 5% dextrose visualized by TEM and
SEM, respectively. Bars, 50 nm.
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Fig. 3.
Transmission electron microscopy of positive
and negative liposomes. Chimeric ONs with or without PEI were
encapsulated in liposomes as described under "Experimental
Procedures." The liposomal formulations were applied to
glow-discharged formvar carbon-coated grids and negatively stained with
a 2% ammonium molybdate solution. a, positive liposomes
were prepared with PEI·ON complexes and extruded to a mean diameter
of 50 nm. The chimeric ON-PEI complexes were 15-20 nm in size as
determined by GEMMA and light scattering. Magnification was × 225,000. b, negative liposomes containing encapsulated naked
chimeric molecules appearing as punctate irregularities. Magnification
was × 112,500; inset magnification was × 200,000. Freeze-fracture analysis of the anionic liposomes
(c) alone or (d) containing encapsulated chimeric
ONs. The mean diameter of the chimeric ON determined by GEMMA was ~4
nm. Magnification was × 112,500; inset magnification
was × 225,000.
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L-PEI was analyzed for sugar content as well as for
unconjugated secondary amines. The conjugation of lactose and PEI
amines was reproducible and increased with incubation time and lactose concentration (Fig. 4). The percentage of
lactose covalently linked to PEI ranged from 24.1 to 25.8% (Table
I), and this agreed with the detectable
levels of free amines. The optimal amounts of L-PEI or PEI
per µg of nucleic acid were calculated based on the nmol of secondary
amines required to completely inhibit nucleic acid detection by
ethidium bromide on an agarose gel (16). The complexed ONs were
resistant to nuclease degradation in contrast to free RNA/DNA ONs,
which were completely degraded (Fig. 5).
Chimeric molecules encapsulated in the different liposome formulations showed a similar protection from nuclease degradation (data not shown).

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Fig. 4.
Lactose conjugation to PEI. 25-kDa PEI
and lactose were conjugated with sodium cyanoborohydride as described
under "Experimental Procedures." After 10 days, the samples were
dialyzed against Milli Q® water for 48 h with 1-2
changes/day. Lactosylation, determined as galactose remaining in the
dialyzed product, increased almost linearly with the concentration of
lactose.
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Fig. 5.
Gel analysis of PEI and L-PEI complexes.
PEI·ON complexes were analyzed on a 4% low melting point agarose
gel. Lane 1, L-PEI·ON, DNase, and RNase
followed by heparin dissociation; lane 2, blank; lane
3, PEI·ON, DNase, and RNase followed by heparin dissociation;
lane 4, 10-base pair ladder; lane 5, PEI·ON
treated with DNase and RNase after heparin dissociation; lane
6, L-PEI·ON treated with DNase and RNase after
heparin dissociation; lane 7, untreated chimeric ONs.
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In Vitro Luc Expression in Hepatocytes Using L-PEI·PGL3 Plasmid
Complexes--
L-PEI (25 kDa) was evaluated for
transfection efficiency using the PGL3 plasmid encoding luciferase
under control of the SV40 promoter. Interestingly, when the sugar:amine
ratio in L-PEI ranged from approximately 0.1 to 1.7, no
significant difference was observed in the level of luciferase
expression at plasmid doses of
2 µg (Table
II). Significant differences in
luciferase expression occurred only at the 1-µg plasmid dose
(p < 0.03) in comparing low level (sugar:amine = 0.051) to high level (sugar:amine = 1.19) lactosylation. Only the
low level L-PEI displayed a clear dose response, even at
the 1-µg dose (Table III). Combining
the L-PEI with naked PEI at a 1:1 molar amine ratio
significantly improved (p < 0.05) the transfection
efficiency over the entire range of lactosylation (Table II).
Complexes were also prepared at different DNA phosphate to
L-PEI/PEI amine ratios using the 25% L-PEI.
The transfection efficiency of PEI with 4 µg of plasmid compared
favorably with the L-PEI/PEI mix at the 1:10
phosphate:amine ratio (Table IV). That
represented an effective mean cation:anion charge ratio of ~2 (28).
However, using 2 µg of plasmid and/or other ratios (1:6 and 1:4,
representing cationic to anionic charge ratios of ~1.2 and neutral,
respectively), the L-PEI/PEI mixes were significantly more
efficient (p < 0.05). At both plasmid doses, the
transfection efficiency of the L-PEI/PEI at a 1:10 ratio
was >1:6, which was >1:4, as previously observed with the nontargeted
PEI (28, 29). The molar ratio of amine derived from either PEI or
L-PEI was also varied to determine the effect on
transfection efficiency with 2 µg of plasmid. Increasing the molar
ratio of amines to 2:1 L-PEI:PEI decreased the luciferase expression >90% at either the 1:10 or 1:6 phosphate to amine ratio (p < 0.001). Alteration of the molar amine ratio to
1:2 L-PEI:PEI also resulted in a significant loss of
luciferase expression (p < 0.05); however, the extent
of the decrease was substantially less at the 1:10 ratio than at the
1:6 ratio, suggesting nonspecific cationic uptake of the plasmid at
this ratio. In fact, when 100 mM D-galactose
was included in the transfections to inhibit receptor uptake, at the
1:10 ratio luciferase expression was decreased only 20% using 2 µg
of plasmid. In contrast, under similar conditions, the 1:6 ratio showed
an 88% decrease (4.8 × 105 ± 7.8 × 104 relative light units/mg of protein) relative to
controls (p < 0.001).
Nuclear Localization of the Chimeric Molecules--
There was
almost no detectable cytoplasmic or nuclear fluorescence with the 1:4
ON-PEI complexes in the primary hepatocytes after 24 h of
transfection. In contrast, both the cationic and anionic liposome
formulations exhibited cytoplasmic and even greater nuclear labeling
with the fluorescent ONs. Furthermore, the nuclear fluorescence with
the anionic liposomes (Fig.
6b) was more intense than with
the cationic liposomes (Fig. 6a). Interestingly, the neutral
liposomes displayed somewhat greater cytoplasmic than nuclear
fluorescence. Co-incubation of all three targeted liposomes with 50 mM D-galactose resulted in significant
inhibition in uptake of the fluorescently labeled ONs (Fig.
6c). Studies with L-PEI indicated that the
targeted system was much more efficient in the intracellular delivery
of the fluorescent chimeric molecules than the unmodified carrier. This
was apparent for both the 800- and 25-kDa polymers (data not shown). In
particular, for the 25-kDa derivative, a 1:6 ratio resulted in
significant nuclear labeling as well as some punctate staining
primarily in the perinuclear regions (Fig.
7c). This was also observed
with the 1:10 phosphate:amine ratio; however, the abundance of punctate
fluorescence in the cytosol and especially in the perinuclear region
was increased (Fig. 7a). Galactose at 100 mM
almost completely inhibited the appearance of the fluorescein label in
cells incubated with the 1:6 phosphate:amine complexes (Fig.
7d). Although some detectable nuclear labeling was still
observed with the 1:10 ratio, almost no fluorescence was present in the
cytosol or perinuclear regions (Fig. 7b).

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Fig. 6.
Confocal micrographs of rat hepatocytes
transfected with liposome-encapsulated fluorescently labeled RNA/DNA
ONs. Cells were transfected with (a) positive and
(b) negative liposome-encapsulated fluorescent chimeric ONs
as described under "Experimental Procedures." After 24 h of
transfection, the hepatocytes were fixed using 4% paraformaldehyde in
phosphate-buffered saline. Postfixation, the cells were counterstained
using the cationic membrane probe DiI to visualize the cytoplasm.
Double-label detection of the DiI fluorescence (orange) and
fluorescein (green) was performed using a Bio-Rad MRC1000
confocal microscope. c, negative liposome-mediated uptake of
fluorescent ONs was inhibited in the presence of 50 mM
D-galactose.
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Fig. 7.
Confocal micrographs of primary rat
hepatocytes transfected with fluorescently labeled RNA/DNA ONs
complexed to L-PEI. 25-kDa PEI was lactosylated as described under
"Experimental Procedures." L-PEI and PEI amines were
mixed at a 1:1 molar ratio and combined with the fluorescent ONs at a
1:10 or 1:6 ratio of ON phosphates to PEI amines prior to transfection
of the isolated hepatocytes. a and b show uptake
of the 1:10 fluorescein-labeled PEI·ON complex with and without 100 mM D-galactose, respectively. c and
d show uptake with the 1:6 complex with and without 100 mM D-galactose, respectively.
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Mutant Conversion of the Rat Factor IX Gene--
Chimeric ONs were
designed that targeted the nontranscribed factor IX genomic DNA strands
(Fig. 8). The RNA/DNA sequence of the
molecules was identical in sequence to the wild-type gene except for
the indicated substitutions in the pentameric stretch of DNA residues
corresponding to the targeted nucleotide at Ser365.
Isolated rat hepatocytes were transfected with the factor IX chimeric
molecules to induce targeted nucleotide exchange of Ser365
Arg365 in the rat genomic sequence. Conversion results
in a gene sequence change from the wild-type A:T pairing to a mutant
G:C pairing, which encodes an inactive form of factor IX.

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Fig. 8.
Factor IX chimeric ON sequence and nucleotide
targeting strategy. The homologous sequence in the rat factor IX
gene is aligned to complement the corresponding chimeric ON sequences.
The Ser365 codon is boxed in gray,
and the targeted nucleotides are underlined. The duplex
chimeric structures are shown with the 2'-O-methylated
modified RNA residues depicted by lowercase; DNA residues are shown in
capital letters with the center mismatched nucleotide pair
indicated in gray shading. The G:C clamp is identified by an
artificial break in the sequence complementary to the 3' to 5' bridge.
Isolated rat hepatocytes were transfected with the factor IX chimeric
ONs designed to induce a single cytidine point mutation at
Ser365 (gray box) within the coding region of
the rat factor IX cDNA. The extent of conversion of a wild-type
adenosine residue to a cytidine at Ser365 was determined by
32P-labeled ON hybridization and DNA sequence analysis as
described under "Experimental Procedures."
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Conversion of the Targeted Nucleotide at
Ser365--
The nuclear localization of the fluorescently
labeled chimeric molecules indicated efficient transfection in the
isolated rat hepatocytes. The cells were then transfected with the
unlabeled factor IX chimeric molecules at comparable concentrations
using PEI, L-PEI, or liposomes as the carrier.
Additionally, vehicle control transfections were performed
simultaneously. The cells were harvested 48 h after transfection
and the DNA isolated and processed for hybridization. The frequency of
the targeted A
C nucleotide conversion at Ser365 was
determined by hybridization of duplicate colony lifts of the
PCR-amplified and cloned 374-nucleotide stretch of exon 8 of the factor
IX gene (5, 38). ON probes corresponding to nucleotides 710-726 of the
cDNA sequence were used to distinguish between the wild-type
365A (5'-AAGGAGATAGTGGGGGA-3') or the converted
365C (5'-AAGGAGATCGTGGGGGA-3') sequence.
The overall frequency of conversion of the targeted nucleotide was
calculated by dividing the number of clones hybridizing with the
365C ON by the total number of clones hybridizing with both
ON probes. Vehicle-transfected cells and those transfected with
unrelated chimeric ONs did not yield any clones that hybridized with
the 365C probe. The A
C conversion in the hepatocytes
transfected with the 25-kDa L-PEI/factor IX chimeric molecules
was dose-dependent, with a maximum exchange frequency of
20.2 ± 1.2%. All three liposomal delivery systems also resulted
in a dose-dependent targeted factor IX conversion in
the primary hepatocytes, with significant increases at 270 nM relative to 180 nM (p < 0.05) and 90 nM (p < 0.001) (Table
V). However, only the cationic and
anionic formulations showed significantly increased conversion at the
180 nM dose (p < 0.05 and
p < 0.01, respectively). Both the negative and
positive liposomal delivery systems were efficient in promoting
conversion; however, at the 180 and 270 nM concentrations,
the neutral liposomes were significantly less efficient for conversion
than the negative liposomes (p < 0.001).
In Vivo Nucleotide Exchange in Rat Liver Using L-PEI--
In
vivo administration via tail vein injection of the 1:6 ON:PEI
complexes was associated with significant conversion (48.7 ± 10.4%) of the targeted nucleotide in the intact liver. Additionally, no difference was noted between animals that received the chimeric ONs,
which targeted the actively transcribed strand versus
nontranscribed strand of the factor IX gene. Moreover, when the
frequency of genomic A
C conversion was determined 18 weeks after
the final injection, no significant difference was noted from that
determined at 1 week.
Phenotypic changes in genomic conversion were determined by the
activated partial thromboplastin time at 3, 18, and 46 weeks after the final injections. At 3 weeks, the treated rats
(n = 9) exhibited factor IX activity ranging from 38.2 to 70.8% (mean, 55.8 ± 11.9%). This was significantly decreased
(p < 0.001) from the vehicle control group
(n = 6), which ranged from 100.1 to 117.4% (mean,
112.7 ± 8.7%). At 18 and 46 weeks, no significant changes in
factor IX activity were detected in the experimental group (mean,
56.2 ± 8.2 versus 57.9 ± 6.3%, respectively)
relative to that of the controls (mean, 111.3 ± 7.9 versus 105.4 ± 5.5%, respectively). At each time
point, the decreased factor IX activity was associated with an ~30-s
increase in activated partial thromboplastin time relative to controls.
Treated rats were also subjected to 70% partial hepatectomy of the
liver and analyzed for alterations in factor IX activity. During the
regenerative period in which the liver mass is restored within ~10
days, at least 95% of remaining hepatocytes replicate in two
synchronous waves (36). The factor IX activity had decreased from a
mean of 53.8% observed at 3 weeks to 37.0% of control values at 49 weeks post-injection/partial hepatectomy. The results suggested that in
the intact liver, the site-directed genomic nucleotide exchange is
replicatively stable and results in a long term phenotypic alteration
in the coagulant activity as measured by the activated partial
thromboplastin time.
Sequence Analysis of the Mutated Factor IX Gene--
Sequencing of
the wild-type and mutated factor IX genes was performed to confirm the
results from the filter lift hybridizations. A minimum of 10 independent clones hybridizing to either 365A or
365C from the intact liver or isolated hepatocytes were
analyzed. The results of the sequencing indicated that colonies derived from the factor IX-transfected primary hepatocytes hybridizing to the
365C ON probe converted to a C at Ser365. The
same A
C conversion at Ser365 was observed in the
clones derived from the transfected rat liver that hybridized with the
17-mer 365C probe. In contrast, those colonies hybridizing
to 365A exhibited the wild-type factor IX sequence. No base
substitution other than the targeted change at Ser365 was
detected in the clones sequenced. Additionally, both the start and end
points of the cloned 374-nucleotide PCR-amplified genomic DNA from the
isolated hepatocytes and intact liver corresponded precisely to those
of the primers used for the amplification process, indicating that the
sequenced DNA was derived from genomic DNA rather than nondegraded
chimeric ONs (data not shown).
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DISCUSSION |
Chimeric RNA/DNA molecules have been successful in promoting
single nucleotide exchange in the rat factor IX gene (5) and in the
-globin gene responsible for sickle cell anemia (4). These ONs, when
combined with a versatile delivery system, could potentially be useful
therapeutic agents in treating certain genetic diseases. Liposomal
delivery systems have long been identified as safe and effective drug
carriers capable of being modified to target various cells and tissues.
The most common liposomes used for gene delivery are those prepared
with various cationic lipids in combination with a fusogenic lipid (15,
40). However, the majority of the cationic lipid formulations tend to
be entrapped in the pulmonary capillaries, thus limiting their access
to other organs (41, 42).
In this study, cationic, neutral, and anionic targeted liposomal
formulations were prepared and characterized, and the efficacy of each
system was assessed for potential use as a delivery system for RNA/DNA
ONs. In contrast to previously reported liposomal formulations of
nucleic acids, the chimeric molecules were directly encapsulated within
the liposomes rather than relying on nonspecific interaction or
association. Dioleoyl trimethylammoniumpropane was chosen as the
cationic lipid because of its decreased toxicity (43) and its more
efficient uptake by liver cells as compared with other cationic lipids
(44-46). Dioleoyl phosphatidylserine was selected as the anionic lipid
due to its efficiency in the delivery of DNA (47) and its somewhat
greater intrahepatic distribution (48, 49). Galactocerebroside was
included in all the formulations, since it has been used successfully
as a targeting ligand for the hepatocyte ASGPR (50).
Light scattering measurements and electron microscopy studies revealed
that the cationic and anionic liposomes remained stable after
encapsulation of the chimeric ONs. Neutral liposomes, however, were
thermodynamically unstable and tended to aggregate within hours of
preparation. Efficient and stable sizing of the anionic and cationic
liposomes to 50 nm was achieved using membrane extrusion. The small
size of the liposomal delivery vehicle is critical, since the access to
the hepatocyte through the fenestrated epithelium of the liver sinusoid
is greatly enhanced when particles are <100 nm in size (51). The
smaller size of the liposome also significantly decreases nonspecific
trapping of the particles in the lungs and spleen (18, 42), while
including the targeting ligand provides hepatocyte-specific uptake via
the ASGPR (18-20, 50). The incorporation of dioleoyl
phosphatidylserine in the anionic formulation may have promoted
hepatocyte uptake, since this lipid constituent appears to
preferentially direct liposomes to hepatocytes in vivo (49).
The ability to encapsulate the naked chimeric ONs in the anionic and
neutral liposomes may have resulted, in part, from their small and very
uniform size (~4.4 nm). Interestingly, the heating and cooling of the
chimeric molecules altered their GEMMA distribution from bimodal to
unimodal. The observed bimodal distribution, with the peaks differing
by the cube root of 2 in the unheated population, resulted from a
dimeric form of the chimeric ON (52). In fact, both monomeric and
dimeric bands were detected when analyzed by gel electrophoresis (data
not shown).
All three liposome preparations were efficient in delivering the
chimeric molecules intracellularly and promoting a significant A
C
conversion at the Ser365 position in the rat factor IX
gene. The in vitro conversion efficiencies obtained with the
different liposomal formulations were consistently greater than
non-L-PEI using similar concentrations of ONs (5). As
previously reported, when targeted to a specific receptor, such as that
for folate, the negatively charged liposomes exhibited better cellular
uptake than their positively charged counterparts (26). This difference
in cellular uptake may account for the increased conversion efficiency
of the targeted site observed with the negative liposome-encapsulated
chimeric ONs, since transfection efficiency and conversion rate
correlate in vitro (22). Additionally, by confocal
microscopy, cells transfected with the cationic liposomes displayed
more punctate cytoplasmic localization of the labeled chimeric
molecules than the anionic transfected cells, suggesting less efficient
release of the ON complexes from the endocytic compartment. While the
cationic and anionic liposome formulations were efficient, nontoxic,
and stable, extrusion of the vesicles to ~50 nm in size often
resulted in encapsulation of 60% or less of the initial concentration
of chimeric molecules.
A number of nonliposomal delivery systems have been developed and
characterized. These formulations typically involved condensing the
nucleic acid with a polycation, thereby compacting the molecules as
well as protecting them from degradation by various nucleases (19, 20,
27, 54). Additionally, the polycations have been modified to promote
receptor-specific targeting. For example, poly-L-lysine
coupled to asialoorosomucoid (19) and galactose (17, 18, 53) markedly
improved delivery of a number of transgenes to hepatocytes by targeting
to the ASGPR. In this study, we modified PEI by attaching
lactose to its amine nitrogens by a simple procedure, thereby targeting
the L-PEI to the ASGPR. This modification
resulted in a significantly improved delivery to the cells.
Interestingly, the 25-kDa L-PEI was more effective than the 800-kDa
L-PEI for in vitro delivery of the chimeric molecules (5),
compared with the unmodified carrier (27). The observed aggregation of
the ON-PEI and L-PEI complexes at high loading doses was
readily inhibited by complexing the molecules in 5% dextrose instead
of 0.15 M NaCl. This simple modification maintains the
complexes as small, discrete particles rather than the network-like
clusters observed in NaCl.
Several ratios of the RNA/DNA phosphate to 25-kDa L-PEI plus PEI amine
were tested for hepatocyte uptake in vitro. Both a 1:6 and a
1:10 ratio demonstrated intense nuclear and some cytoplasmic labeling
with the fluorescent ONs. However, uptake of the molecules by the
ASGPR was completely inhibited in the presence of 100 mM D-galactose only at the 1:6 ratio. These
data suggest that the decreased positive charge ratio of the 1:6 ON
phosphate to amine induces binding by the ASGPR, while the
increased cationic nature of the 1:10 complexes promotes
internalization of the labeled ON complexes by both receptor-mediated
as well as nonspecific pathways. This is supported by the data from the
PGL3 plasmid studies, since the luciferase activity was markedly
decreased only with the 1:6 phosphate:amine ratio in the presence of
100 mM D-galactose. Additionally, PEI
lactosylation (sugar:amine ratio) of 0.1 or higher significantly
increased delivery of the luciferase reporter plasmid PGL3 to isolated
rat hepatocytes. Combining the L-PEI and PEI improved the
transfection efficiency significantly over L-PEI alone, and
a 1:1 molar ratio of amines from each form of PEI was determined to be
optimal. The decreased transfection efficiency observed with the
L-PEI alone may be due to slightly inefficient compaction
of the plasmid DNA by the L-PEI. In fact, the degree of
compaction of plasmid DNA by PEI, rather than ionic charge, directly
affects the cytoplasmic to nuclear translocation of plasmid DNA (54).
The efficiency of the 25-kDa L-PEI and PEI mix to promote expression of
the PGL3 plasmid encoded luciferase in the nonreplicating rat
hepatocytes may be due, in part, to the ability of this cation to
promote efficient delivery of the plasmid DNA from the cytoplasm to the
nucleus. When injected directly into the nucleus, the complex of PEI
with a plasmid did not prevent expression of the encoded DNA (54).
However, our preliminary studies using fluorescently labeled
L-PEI and the chimeric molecules suggest that PEI does not
enter the nucleus with the RNA/DNA ONs (data not shown).
The ultimate success of any gene therapy approach requires prolonged
systemic expression of therapeutic levels of the delivered transgene.
Although modification of hepatic gene expression using replication-deficient adenoviruses has been successful, there continues
to be significant immunogenicity and lack of sustained expression of
the transgene in the absence of repetitive treatments (55-57). The
alternative approach provided by the RNA/DNA technology, i.e. repairing a liver genetic defect in situ by
site-specific nucleotide exchange, may significantly overcome some of
these challenges. To this end, the in vivo results with the
25-kDa L-PEI·ON complexes demonstrated that a mean 48% A to C
conversion in the genomic DNA for the rat factor IX gene in liver could
be achieved using this system. This genomic sequence alteration
produced a phenotypic change resulting in a ~45% decrease in factor
IX coagulant activity. Moreover, the phenotypic expression of this
targeted genomic base exchange was still observed 46 weeks after the
injection of the chimeric molecules. Finally, the genomic A to C
conversion induced by the chimeric molecules is replicatively stable as
demonstrated by the lack of significant change in the phenotypic
expression of this trait following partial hepatectomy. In support of
these observations, RNA/DNA ONs were recently shown to correct a
point mutation in the tyrosinase gene of melanocytes derived from
albino mice (58). Those results demonstrated a permanent and stable gene correction by clonal analysis, restoring enzyme activity and
melanin production.
In conclusion, three liposomal systems and a novel ligand-based carrier
were developed for targeted gene delivery to the ASGPR in
liver. Each system was effective in mediating nucleotide exchange in
genomic DNA with the RNA/DNA chimeric molecules. Anionic liposomes and
a 25-kDa lactosylated PEI product were the most efficient vehicles
in vitro. The latter was also shown to be highly effective for in vivo delivery, which may in part be due to its
ability to promote cytoplasmic to nuclear translocation of nucleic
acids in nonreplicating cells (54). In a separate study, we have
corrected the genetic mutation for the UDP-glucuronosyltransferase-1
gene in the Gunn rat model of Crigler-Najjar syndrome type I. The
results obtained in vivo with both L-PEI and
anionic liposomes were essentially identical, indicating that both
systems are equally effective for delivery of the chimeric ONs to
hepatocytes (59). The ease with which these systems can be prepared and
characterized makes them attractive formulations for therapeutic
delivery of gene products.