ABSTRACT
Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer
characterized by a branched spherical shape and a high density surface charge.
We have investigated the ability of these dendrimers to function as an
effective delivery system for antisense oligonucleotides and `antisense
expression plasmids' for the targeted modulation of gene expression. Dendrimers
bind to various forms of nucleic acids on the basis of electrostatic
interactions, and the ability of DNA-dendrimer complexes to transfer
oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed
in an
in vitro
cell culture system. Cell lines that permanently express luciferase gene were
developed using dendrimer mediated transfection. Transfections of antisense
oligonucleotides or antisense cDNA plasmids into these cell lines using
dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused
~25-50% reduction of baseline luciferase activity. Binding of the
phosphodiester oligonucleotides to dendrimers also extended their intracellular
survival. While dendrimers were not cytotoxic at the concentrations effective
for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be
effective carriers for the introduction of regulatory nucleic acids and
facilitate the suppression of the specific gene expression.
For over 20 years, antisense inhibition of translation has been considered an
effective technique for the modulation of gene expression (
1
). The general principle of antisense inhibition, whether employing synthetic
oligonucleotides or antisense expression plasmid systems, relies on the
specificity of Watson-Crick base pair formation (
2
,
3
). This enables complementary nucleic acid sequences to target and inhibit steps
in the transcription and translation of specific genes (
4
). The ability of antisense techniques to down-regulate the expression of specific genes is well documented in both
in vitro
and
in vivo
systems (
5
,
6
). However, given the inefficiency of naked DNA entry into the cells, most of
these studies have required micromolar concentrations of oligonucleotides with
modified DNA structures (to increase the availability and the stability of the
nucleic acid) in order to obtain a significant inhibitory effect (
7
). These requirements limit the utility and the potential therapeutic efficacy
of this approach.
Several methods that enhance the transfer of DNA into eukaryotic cells offer the
potential to facilitate antisense applications. Cationic lipid preparations
have been demonstrated
in vitro
to improve the effectiveness of antisense oligonucleotides, presumably through
increased transfer into cells (
8
,
9
). However, the currently available lipid preparations are not effective and
have cytotoxic effects
in vivo
that often make it difficult to determine specific antisense effects (
9
). Other techniques for gene transfer rely on adenoviral and retroviral vectors
(
10
,
11
) and may supply a means for transfer of antisense expression sequences
incorporated into the viral genes. Unfortunately, these methods cannot be
employed with synthetic oligonucleotides and pose problems with immunogenicity
and cell targeting
in vivo
. Despite these difficulties, it seems likely that improving the efficiency of
transfer of antisense oligonucleotides and targeting these agents to particular
cells is crucial to the success of antisense technology.
PAMAM dendrimers are a new type of polymer with a molecular architecture
characterized by regular, dendric branching with radial symmetry (
12
). This results in dendrimers having unusual physical and chemical properties.
PAMAM dendrimers are currently the only class of dendritic macromolecules that
are reliably produced in large quantities and that can be precisely synthesized
over a broad range of molecular weights similar to that of proteins. These
molecules range in size from 10 to 100 Å, with each generation, or layer, of the polymer adding ~10 Å to the diameter of the molecule (Fig.
1
). The number of surface primary amino groups doubles with each generation,
reaching 4096 for a tenth generation dendrimer (
12
,
13
). The defined structure of these molecules and their large number of surface
amino groups has led to dendrimers being employed as a substrate for the
attachment of antibodies, contrast agents and radionucleotides for applications
in a number of different areas of biology and medicine (
14
-
16
). Studies using antibody/dendrimer conjugates
in vitro
and
in vivo
in experimental animals have documented these conjugates to be non-toxic and able to target biologic agents to specific cells (
14
,
15
).
The synthesis of Starburst PAMAM dendrimers has been previously described in
detail (
13
,
17
) and is presented schematically in Figure
1
.
The luciferase reporter plasmid pCMVLuc was constructed using pcDNAI vector
(Invitrogen) and
Hin
dIII-
Bam
HI fragment, containing the entire luciferase cDNA and SV40 polyadenylation
signals of pGL-basic (Promega). Two types of antisense luciferase mRNA expressing plasmids were generated. The pCMVAsLuc, containing the ATG region was constructed by ligating the
0.66 kb
Hin
dIII-
Eco
RI fragment of luciferase gene (from pGL-basic) with
Bam
HI digested pcDNAI vector. The fragments were made compatible by filling in with
Klenow polymerase (
18
). Transformants were screened using diagnostic digests with
Hin
dIII and
Nar
I to identify insert orientation, and sequenced to confirm insertion sites of
the truncated luciferase gene. The pCMVAsLuc[part]ATG was generated by
Xba
I digest that deletes 48 nucleotides of the luciferase coding sequence of
pCMVAsLuc. The pGEM-Luc (Promega) and pCMV[beta]gal (
19
) (Clontech) were purchased from the suppliers.
Plasmid DNA was amplified in bacteria and then isolated by double cesium
chloride gradient (
18
) to ensure the purity of the DNA preparation. Dendrimers were diluted to an
appropriate concentration in buffer containing 20 mM HEPES pH 7.9, 100 mM KCl,
0.2 mM EDTA, 0.5 mM DTT and 20% glycerol, all solutions stored at 4oC until required. DNA-dendrimer complexes were formed by incubating the two components
together in 15-50 [mu]l water for a minimum of 5 min at room temperature. Ratios of nucleic
acid to dendrimer were based on the calculation of the electrostatic charge
present on each component; the number of phosphate groups in the nucleic acid
versus the number of terminal NH
2
groups on a dendrimer. For example, given that the number of bases in 1 [mu]g DNA is 1.71 * 10
15
, ~1.71 * 10
15
negative charges are present
per 1 [mu]g DNA while a G7 (NH
3)
dendrimer has ~2.65 * 10
15
charges per microgram. Therefore, to obtain a 1:1 charge ratio, 1 [mu]g DNA was mixed with 0.65 [mu]g dendrimer.
A number of stable clones that demonstrated the long-term expression (4-6 months) of transfected luciferase cDNA were generated from D5
mouse melanoma and Rat2 embryonal fibroblast cell lines. Cells were transfected
with G7 (NH
3
) dendrimer and pCMVLuc plasmid DNA. Clones with integrated plasmid DNA were
initially selected by incubation in neomycin (G418, Geneticin, Life
Technologies) at 300 [mu]g/ml for 3 weeks. The numbers of G418-resistant clones were determined by visually counting the colonies of
cells after staining with either eosin or methylene blue. Neomycin resistant
clones were assayed for luciferase expression by chemiluminescence as described
below. Presence of integrated pCMVLuc DNA was confirmed by Southern
hybridization of chromosomal DNA using a fragment of the luciferase gene as a
probe.
Rat2 cells were maintained in D-MEM medium (Gibco-BRL) with 5% Nu-serum (Collaborative Biomedical Products), 1% penicillin-streptomycin and 2 mM L-glutamine. D5 cells were cultivated in CM medium
RPMI 1640 with 10% fetal calf serum, 1% penicillin-streptomycin, 2 mM L-glutamine, 50 [mu]M 2-mercaptoethanol, 1 mM non-essential amino acids (Life Technologies). U937
human histiocytic lymphoma cells were maintained in RPMI 1640 with 5% fetal
calf serum, 1% penicillin-streptomycin. All cells were incubated at 37oC in 5% CO
2
.
A 27 base single-stranded phosphodiester oligonucleotide 5'-GGCGTCTTCCATTTTACCAACAGTACC-3', complementary to the ATG region of pCMV-Luc plasmid (antisense-ATG oligo) and its complement,
identical to the coding strand of the ATG region of luciferase gene (sense-ATG oligo), were synthesized using 394DNA/RNA Synthetizer (Applied
Biosystems) and purified by HPLC at the Biomedical Research Core Facility of
the University of Michigan.
Transfections with dendrimers were performed and analyzed using assays for
luciferase activity reflecting the luciferase gene expression. DNA expression
plasmids pCMV-Luc, or antisense expressors pCMVAsLuc and pCMVAsLuc[part]ATG as well as single-stranded oligonucleotides were complexed with dendrimers as
described above. DNA (50-1000 ng DNA per well of transfected cells) was mixed
with dendrimers at a variety of charge ratios, from 1:10 to 1:50 and were then
allowed to complex for >= 5 min at room temperature. Twenty-four-well plates, seeded 24 h before the transfection with ~2 * 10
4
cells per well were washed once with serum-free media. Fifteen microliters of the solution containing 50-500 ng DNA-dendrimer complexes were added to 185 [mu]l serum free-media in each well of cells and incubated for 3 h
at 37oC. The serum-free medium containing the complexes was then washed out of the
cells, and standard growth media was substituted. The cells remained in culture
for 24-96 h before being harvested to analyze for expression of luciferase.
A single-stranded 27 base long oligonucleotide was radiolabelled with [[gamma]-
32
P]ATP using T4 polynucleotide kinase. After separation from unincorporated
label, ~5 ng radiolabelled DNA (2 * 10
6
c.p.m.) was mixed with 100 ng cold oligonucleotide and complexed with G5, G7
and G9 EDA core dendrimers in a 1:5, 1:10 and 1:100 DNA to dendrimer charge
ratio. U937 cells were washed twice with serum-free media and plated at 2 * 10
5
cells/well in 24-well plates. Serum-free medium (0.2 ml) containing radioactive oligonucleotides or
oligonucleotide-dendrimer complexes was added. The DNA was incubated with the cells for 4
h. The cells were washed with serum-free medium, 3 ml complete medium was added and cultures were incubated
for 12, 24, 48 and 96 h. Total nucleic acids were isolated from cells with DNA-zol (MRC Inc., Cincinnati) with an additional phenol-chloroform extraction
and precipitation with 5 vol EtOH in the presence of 0.3 M NaOAc. Precipitates
were then washed twice with 80% EtOH at 4oC. All samples were resuspended in 20 [mu]l buffer containing 0.5% SDS, 1 mM EDTA, 10 mM Tris-HCl pH 7.8 and radioactivity was quantified using a
scintillation counter.
Luciferase expression was
quantified in lysates of transfected cells at 24 h after transfection by
measuring the light emission resulting from 10 [mu]l cell lysate incubated with 2.35 * 10
-2
[mu]mol of luciferin substrate (Promega, Technical Bulletin
No.101). Light intensity was measured in a
chemiluminometer (LB96P; EG&G Berthold), and adjusted according to the total protein concentration of the
sample. The total protein concentration in the cell lysate was measured in a standard
protein
assay (DC protein assay; BioRad, Richmond, CA). Expression of luciferase was
also examined using a coupled transcription-translation system (TNT Coupled Reticulocyte Lysate System; Promega)
containing 200 ng luciferase expression plasmid (either pCMV-Luc or pGEM-Luc). Luciferase activity was determined as previously described.
Statistical analysis was performed using Systat 5.2 software for Macintosh.
Errors were calculated as standard deviations and differences between samples
were analyzed by ANOVA.
The development of a system to evaluate the efficiency of the transfer of
antisense regulatory DNA using Starburst PAMAM dendrimers began with an
analysis of the specific effect of antisense oligonucleotides on
in vitro
transcription-translation of luciferase in a cell-free expression system. Increasing amounts (50 ng-1.0 [mu]g) of the antisense-ATG oligonucleotide were added to the coupled
transcription-translation system containing 200 ng of either pCMV-Luc (Fig.
2
A) or pGEM-Luc (Fig.
2
B) as templates and suppressed luciferase expression in dose-dependent fashion to 1.5% of baseline control level (98.5% inhibition,
Fig.
2
A). In contrast, identical concentrations of sense oligonucleotides had no
effect on the luciferase expression. High concentrations of dendrimers (130 [mu]g/ml) caused minimal non-specific suppression of luciferase synthesis in the coupled
transcription-translation reaction, but did not interfere with the specific inhibitory
effect of the antisense oligonucleotide. The expression of a control gene ([beta]-galactosidase from pCMV[beta]gal plasmid) was not altered by the presence of either the
sense or antisense oligonucleotide (data not shown). The sequence specificity
of inhibition was additionally confirmed by the expression of luciferase from a
pGEM-Luc plasmid. This plasmid differs from pCMV-Luc by 6 base pairs in the plasmid 5' of the initiating ATG of the luciferase cDNA, creating a six
base mismatch with the antisense oligonucleotide designed for pCMV-Luc. Expression from pGEM-Luc was inhibited by the pCMV-Luc antisense oligonucleotide to a lesser degree than
expression from pCMV-Luc (Fig.
2
B).
Antisense technology has been proposed as one means for modulating the
expression of specific genes (
20
,
21
). The mechanism for this is thought to be the formation of heteroduplexes with
the mRNA for a specific protein that either prevent the translation of the
message or accelerate the degradation of the mRNA (
22
). One of the major problems with the use of antisense regulatory nucleic acids
is the difficulty in achieving functional concentrations of oligonucleotides in
cells and the rapid degradation of these molecules by cellular nucleases. The
low efficiency of oligonucleotide cellular uptake is usually compensated
through the use of very high concentrations of oligonucleotides, while chemical
modifications to the phosphodiester bond improve stability of the genetic
material (
23
,
24
). The efficiency of antisense nucleic acids can be greatly enhanced when
delivered into cells using transfecting agents. Cationic liposome-mediated transfection of 100 nM phosphorothioate antisense ICAM-I oligonucleotide resulted in 90% reduction of protein synthesis in
endothelial cells (
25
), and 50-80% reductions of human protocollagenase mRNA expression in fibroblasts
by 200 nM phosphorothioate specific oligonucleotides (
26
). However these transfer techniques have proved problematical in delivering
oligonucleotides
in vivo.
As a result, the practical application of antisense technology awaits more
efficient delivery systems.
Our studies indicate that Starburst dendrimers can be used as a transfection
reagent for the delivery of antisense oligonucleotides and plasmid expression
vectors coding antisense mRNA. Inhibition of the expression of the reporter
luciferase gene by antisense oligonucleotides as well as antisense mRNA ranged
from 30 to 60% of the control depending on the DNA concentration, type of
dendrimer used and charge ratios of DNA-dendrimer complexes. In contrast to the nanomolar or micromolar
concentrations of oligonucleotides needed for inhibition reported by others (
25
-
27
), we have achieved specific inhibition of targeted gene expression with
picomolar concentrations of specific oligonucleotides when delivered by
dendrimers. Sequence specificity of inhibition was documented by using sense
control oligo- nucleotides and a derivative of the antisense luciferase expression
plasmid that lacked the ATG region of luciferase gene. The efficiency of
transfer was aided by the finding that dendrimers were not toxic to cells in
the concentrations required for gene transfer. We have failed to obtain
consistent effects on luciferase expression in Jurkat, Rat2 and D5 cells when
antisense oligonucleotides were transfected with liposomes, predominantly due
to cytotoxicity. This suggests that complexing DNA with dendrimers results in
highly efficient oligonucleotide delivery and avoids sequence-independent, carrier-mediated alterations on cellular function (
28
-
32
), and can potentially lead to
in vivo
applications of regulatory nucleic acids, that are not possible with the
currently-available delivery techniques (
33
,
34
).
Stability of oligonucleotides is a necessary requirement for the application of
antisense technology to inhibit gene expression
in vivo
. This often precludes the use of `naked' phosphodiester oligos because of
either degradation in serum or intracellular destruction by endosomes or
nucleases (
8
,
9
). Significant efforts have been made toward the development of nuclease-resistant oligonucleotides; in particular phosphorothioates and
methylphosphonates (
35
,
36
). From our studies, phosphodiester oligonucleotides appear to have the
increased stability when complexed to dendrimers. This may allow the use of non-modified oligonucleotides in place of costly and sometimes toxic modified
phosphoderivatives (
37
). In addition, it is of interest that oligonucleotides bound to dendrimer
appear to function as specifically and effectual as free oligonucleotides. It
is possible that the binding of the oligonucleotide phosphate backbone to the
dendrimer surface does not interfere with the ability of the bases to form
hydrogen bonds with the complementary sequences
.
Antisense nucleic acids (oligonucleotides and plasmids coding antisense mRNA)
show great promise for the specific inhibition of gene expression. Our studies
indicate that the complexing of oligonucleotides with dendrimers increases
cellular uptake and prolongs the activity of regulatory sequences while not
altering the functional capability of those molecules. Application of
dendrimers as a delivery system for nucleic acid molecules may lead to
improvements in this technology and may facilitate the development of
therapeutic antisense techniques.
This studies presented in this manuscript were supported by grants from the
Pardee Foundation and Dendritech, Inc., as well as R43 CA 68820.
REFERENCES
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