Nuclear entry of nonviral vectors
DA Dean1, DD Strong2and WE Zimmer3
1Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA;
2V A Loma Linda Health Care System, Loma Linda, CA, USA; and3Department of Medical Pharmacology and Toxicology,
College of Medicine, Texas A&M University, College Station, TX, USA
Nonviral gene delivery is limited to a large extent by multiple
extracellular and intracellular barriers. One of the major
barriers, especially in nondividing cells, is the nuclear
envelope. Once in the cytoplasm, plasmids must make their
way into the nucleus in order to be expressed. Numerous
studies have demonstrated that transfections work best in
dividing populations of cells in which the nuclear envelope
disassembles during mitosis, thus largely eliminating the
barrier. However, since many of the cells that are targets for
gene therapy do not actively undergo cell division during the
gene transfer process, the mechanisms of nuclear transport
of plasmids in nondividing cells are of critical importance. In
this review, we summarize recent studies designed to
elucidate the mechanisms of plasmid nuclear import in
nondividing cells and discuss approaches to either exploit
or circumvent these processes to increase the efficiency of
gene transfer and therapy.
Gene Therapy (2005) 12, 881–890. doi:10.1038/
sj.gt.3302534; Published online 28 April 2005
Keywords: nuclear import; nuclear envelope; plasmid; nuclear localization signal; nonviral gene transfer
The success of nonviral gene therapy has been largely
limited by inefficient gene delivery due to the presence of
multiple extracellular and intracellular barriers to gene
transfer. Perhaps, the major barrier that has not been
successfully overcome is the nuclear envelope. Although
the molecular mechanisms of the nuclear import and
export of proteins, mRNAs, snRNPs, and ribosomes have
been studied in detail over the past 20 years, the
mechanisms of plasmid nuclear import have received
much less attention. It is clear that unless plasmids can
enter the nucleus, they cannot be transcribed. Thus, just
as entry into the cytoplasm across the plasma or
endosomal membrane is a prerequisite for gene transfer,
transport across the nuclear membrane is equally vital.
Nuclear envelope as a barrier to gene
In 1980, Mario Capecchi demonstrated that when
pBR322-based plasmids were injected into the nuclei of
mouse fibroblasts, expression could be detected in about
50–100% of cells, but when the same number of plasmids
were injected into the cytoplasm, no expression was
detected in any of the 1000 injected cells.1Similar
microinjection experiments by Graessman et al2showed
that when between 1000 and 3000 copies of a plasmid
were delivered to the cytoplasm, the level of gene
expression detected was less than 3% of that found
when the DNA was microinjected into the nucleus.
Zabner et al3also found that in Xenopus oocytes, the same
trend was true: whereas DNA microinjected into the
nucleus produced robust gene expression, cytoplasmic
injection resulted in very little expression. Other experi-
ments in a number of mammalian cell types have
confirmed these findings.4–7
Implicit in all of these experiments, and the conclusion
that the nuclear envelope is a barrier to gene transfer, is
the assumption that the cells studied were nondividing.
Indeed, it is known that when cells undergo mitosis, the
nuclear envelope breaks down and the permeability
barrier to the nucleus is lost. Thus, if plasmids (or viral
genomes) are present in the cytoplasm, they can enter the
‘nucleus’ when the envelope is disrupted during this
stage of the cell cycle. For example, it has long been
known that in contrast to lentivruses such as HIV, the
reverse-transcribed genomes of oncoretroviruses cannot
enter the nucleus to integrate and express unless the cells
go through mitosis.8–10Similarly, it is well appreciated
that nondividing or growth-arrested cells cannot be
easily transfected by almost any method. By contrast,
cells undergoing mitosis are much more receptive to
transfection. In one study using primary human airway
epithelial cells, nondividing cells, as measured by lack of
BrdU incorporation, were only 10% as likely to express
gene product as their dividing counterparts.11More
recent studies using synchronized cells have shown that
cells transfected with various lipoplex formulations,
between 50- and 3000-fold more gene product when
transfected in the G2 or G2–M stage as compared to
those transfected in G1.12,13
Published online 28 April 2005
Correspondence: Professor DA Dean, Division of Pulmonary and Critical
Care Medicine, Fienberg School of Medicine, Northwestern University,
240 E Huron Ave, McGaw 2336, Chicago, IL 60611, USA
Gene Therapy (2005) 12, 881–890
& 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00
Regardless of whether the cells undergo mitosis or not,
the levels of DNA that reach the nucleus are low at best.
It has been estimated that following lipoplex-mediated
transfection, between 2000 and 10 000 plasmids are
delivered per cell.14–16Using quantitative PCR, Southern
blot analysis, or electron microscopy, between 1 and 10%
of the plasmids were found in the nuclei of the cells at up
to 24–36 h following DNA addition.6,17In another study
in which plasmids were fluorescently labeled randomly
throughout the DNA and quantified in cells by fluores-
cence activated cell sorting, the numbers were much
higher, with between 30 and 60% of the intracellular
DNA being in the nuclear compartment, depending on
the cell type used.16This suggested that different cells
have differing capacities for DNA nuclear localization.
However, in this study, the modification of the DNAwith
fluorophores every 22 bp may have resulted in plasmids
that are not completely representative of native DNA,
and thus these numbers may be an overestimation.18,19
Regardless, these results demonstrate that a significant
proportion of the DNA that enters the cytoplasm never
arrives in the nucleus. In studies from our lab, increasing
concentrations of expression plasmids were micro-
injected into the cytoplasm or nucleus and 24 h later,
after the synchronized cells had divided, the levels of
gene expression were quantified. We found that it took
30–100 times more plasmid injected into the cytoplasm
compared to the nucleus to give equivalent levels of gene
expression, suggesting that even in dividing cells, the
amount of DNA that gets to the nucleus is low.7Similar
studies from Jon Wolff’s group later confirmed these
findings and illustrate that nuclear import of DNA is a
relatively inefficient process, even when the nuclear
envelope breaks down.19
One major reason that the majority of cytoplasmic
plasmids fail to reach the nucleus is the presence of
cytoplasmic nucleases that act to degrade the ‘free’ DNA.
Clearly, following endocytosis, a significant percentage
of internalized DNA is targeted to the lysosomal
compartment, where it will be degraded. However,
once DNA is freed into the cytoplasm, it is still subject
to degradation prior to nuclear entry. Studies from
Lechardeur et al20showed that single- and double-
stranded plasmids are degraded in the cytoplasm of
of between 50 and 90 min. Similarly, Pollard and co-
demonstrated that this nuclease activity
was calcium-dependent and degraded DNA in cells
with a half-life of less than 2 h. By contrast, several
other studies have not detected such high rates of
degradation, although it is clear that some degree
of DNA disappearance is occurring.22,23For example,
developed mechanistic models for nonviral gene deliv-
ery and their experimental findings suggest that cyto-
plasmic DNA may be degraded at rates between 30 and
1400 molecules per min, depending on the total amount
of input DNA in the cell.15,16,24While these rates seem
high, they would translate to a cytoplasmic half-life of
roughly 5 h. Regardless of the exact rate of degradation,
it is clear that there is a competition between DNA
degradation in the cytoplasm and efficient nuclear
import; if the DNA persists too long in the cytoplasm,
there will be less of it around to enter the nucleus and
lead to gene expression.
Intracellular trafficking of ‘naked’ DNA
The function of the cationic lipid and polymer compo-
nents of transfection complexes is to condense the DNA,
protect it from degradation, and to promote association
with the plasma membrane so the DNA can interact with
and enter the cell. Following endocytosis, in order for the
DNA to enter the nucleus, it must escape the endosome
and become ‘freed’ into the cytoplasm. One question
surrounding this and the nuclear entry of the DNA is
whether the DNA dissociates from the lipoplex or
polyplex complexes prior to or after nuclear entry.
Experiments have been performed that support both
possibilities, but the preponderance of data suggests that
following lipoplex-mediated transfections, the DNA that
drives gene expression in the nucleus is likely free of
lipid. Zabner et al3demonstrated that when liposome-
complexed plasmids were injected directly into the
cytoplasm or nucleus at the same lipid:DNA ratios that
gave optimal transfection efficiency, no gene expression
could be detected. By contrast, certain polyplex com-
plexes, such as PEI, may remain complexed with the
DNA after transport into the nucleus: when PEI–DNA
complexes were microinjected into the cytoplasm or
nucleus, gene expression was detected in either case.25–27
Regardless of whether the complexes dissociate in
the cytoplasm or nucleus, it is unlikely that the DNA
remains ‘free’ for very long. The cytoplasm and nucleus
are filled will numerous DNA-binding proteins, poly-
amines, and other polycations that will complex with the
released DNA. These associations will likely neutralize
the charge on the DNA, condense the DNA, and thereby
reduce the size of the plasmid to possibly aid in nuclear
transport. They may also prevent or promote cyto-
plasmic degradation of the DNA. Further, some of these
proteins may actually mediate the specific nuclear
import of the plasmids.
Plasmid nuclear entry in nondividing cells
Wolff and co-workers first demonstrated that plasmids
can be transported into the nuclei of nondividing cells
via the nuclear pore complex (NPC).28All macro-
molecular traffic that enters or exits the intact nucleus
occurs through the NPC.29They showed that when
microinjected into the cytoplasm of cultured myotubes,
plasmids localized to the nucleus, based on detection
of reporter gene expression, in a dose- and energy-
dependent process. Further, coinjection of agents that
block transport through the NPC (either wheat germ
agglutinin or an antibody against NPC components) also
blocked plasmid nuclear entry and gene expression.
Using a similar microinjection approach, but using in situ
hybridization to detect the injected DNA directly, our lab
later confirmed that plasmids enter the nucleus via the
NPC, but do so in a sequence-specific manner.30When
the 5243 bp SV40 genome was microinjected into the
cytoplasm of a variety of growth-arrested cell types, it
localized to the nucleus within 6–8 h in the absence of
cell division. Import was inhibited by wheat germ
agglutinin, energy-depletion, and antibodies against the
NPC. However, in attempts to compete for import using
a second plasmid type, it was found that import was
sequence specific. Whereas the SV40 genome localized to
Nuclear entry of nonviral vectors
DA Dean et al
the nucleus in the absence of cell division, plasmids
lacking SV40 sequences, such as pBR322, pUC19, or
pGL3-basic, remained in the cytoplasm. This was most
striking when cells were coinjected with SV40 DNA and
one of these other plasmids; the SV40 in situ signal was
nuclear, but the other plasmid’s signal was completely
cytoplasmic. By cloning various sequences from the SV40
genome into pUC19 or pBR322, it was found that the
SV40 enhancer was all that was necessary for this DNA
nuclear import.7,30This sequence and other sequences
supporting DNA nuclear import were termed DNA
nuclear targeting sequences, or ‘DTS’.
DNA nuclear targeting sequences (DTSs)
The DNA sequence from the SV40 genome that
supported nuclear import of an otherwise cytoplasmi-
cally localized plasmid contained the 72 bp enhancer
repeat.7,30When as little as one copy of this enhancer was
cloned into pBR322, the resulting plasmid localized to
the nuclei of microinjected, nondividing cells with the
same kinetics as the full-length SV40 genome with
import first being detected within 40–60 min of injection
(Figure 1). Such sequence-specific DNA nuclear import
has been observed in all mammalian cell types tested to
date, including primary cells and cell lines from mice,
rats, chickens, hamsters, monkeys, and humans.7,30–33In
support of these findings, Greassman et al2demonstrated
that the 72 bp SV40 enhancer lead to increased transcrip-
tion of a herpes TK promoter-driven gene in actively
dividing cells, compared to plasmids lacking the
enhancer, confirming the role of the sequence as an
enhancer. However, when he microinjected the plasmids
into the nucleus or cytoplasm and followed expression,
he found that the enhancer-containing plasmid was more
efficient at stimulating gene expression when the DNA
was microinjected into the cytoplasm than when it was
delivered to the nucleus, suggesting that the classical
‘enhancer’ activity was not the only function of this
sequence. More recent work from other labs has
supported these findings.34,35
Based on the sequence required for DNA nuclear
import, a model was developed to account for the
sequence-specificity (Figure 2). The SV40 enhancer
contains binding sites for a number of ubiquitously
expressed, general transcription factors, such as AP1,
AP2, AP3, NF-kB, Oct1, TEF-1, etc.36Normally, combina-
tions of these transcription factors would bind to the
SV40 enhancer when the genome is in the nucleus to
regulate gene expression during the infectious cycle.
However, since transcription factors, like other proteins,
are translated in the cytoplasm, it is possible that they
could bind to their binding sites on the SV40 enhancer-
containing plasmid to create a protein–DNA complex.
Further, since transcription factors function in the
nucleus, they contain nuclear localization signals (NLSs)
within their amino-acid sequences to direct nuclear
import via interactions with the importin machinery
and the NPC. Normally these transcription factors would
be translated in the cytoplasm, transported into the
nucleus using their NLSs and the importin proteins, and
then bind to their target sites on various promoters and
enhancers within the nucleus. However, if they first bind
to a cytoplasmic SV40 DTS-containing plasmid, the
plasmid could become coated at one or more sites with
a number of proteins that harbor NLSs, which could in
turn bind to the importins. This large multiprotein–DNA
complex could then be carried into the nucleus by the
classic signal-mediated NPC pathways. Thus, the SV40
DTS can be seen as a scaffold for transcription factors
and their bound importin family members, which results
in nuclear import of the entire protein–DNA complex.
Similar mechanisms for nuclear import of the reverse-
transcribed HIV preintegration complex, and other viral
genomes have been suggested.10,37However, the main
difference here is that the SV40 DTS functions to promote
plasmid nuclear import using only endogenous cellular
proteins in the absence of any viral component.
Based on this model for plasmid nuclear import,
it is possible that any eukaryotic promoter, enhancer,
insulator, or regulatory sequence could act as a DTS.
However, this is not the case. A number of strong viral
and cellular promoters have been tested for their ability
to promote nuclear import of plasmids following
transfection or cytoplasmic microinjection, and to date,
most sequences have no such activity. For example, the
CMV immediate early promoter/enhancer, the herpes
TK promoter, and the RSV LTR promoter, all of which are
as robust for transcription as the SV40 early promoter/
enhancer and contain multiple binding sites for a
number of similar transcription factors, have no DTS
activity.2,7,32Further, the presence of multiple transcrip-
tion factor binding sites is not sufficient to create nuclear-
localizing plasmids. How then is the SV40 DTS active for
import but many of these other sequences are not?
First, not all transcription factors can mediate or
participate in DNA nuclear import. In order for a
transcription factor to act as an adapter between the
DTS scaffold and the importin machinery, it must have
functionally and spatially distinct NLS and DNA-
binding domains. Sp1, for example, contains an NLS
that is buried within the zinc-finger DNA-binding
domain.38Under normal circumstances, the NLS is
recognized by the importins, Sp1 is transported into
the nucleus, and then it binds to DNA. However, if it
were to bind to the SV40 promoter/enhancer (which it
does) in the cytoplasm, the NLS would not be accessible
to the importins. In an elegant set of studies, Chan and
Jans39,40demonstrated that although the GAL4 transcrip-
tion factor from yeast can bind to the GAL4 UAS when
present on a plasmid and can interact with high affinity
with importina, it cannot do both at once. Thus, contrary
to initial hopes, GAL4 did not enhance nuclear import
of plasmids or gene expression. By contrast, the NLS of
Figure 1 Plasmid nuclear import in nondividing cells. Plasmids were
labeled using a Cy5-PNA clamp and microinjected into the cytoplasm of
TC7 cells as previously described.7,43The DNA can be seen to begin to
enter and accumulate in the nucleus as early as 40 min following
cytoplasmic injection. If the cells are allowed to incubate for longer times,
up to 100% of the PNA-labeled DNA is detected within the nucleus.
Arrows indicate nuclei in two injected cells.
Nuclear entry of nonviral vectors
DA Dean et al
NF-kB is far removed from the DNA-binding domain of
the protein and could be involved in importin recogni-
tion and nuclear import of the DNA complex.41,42
The presence of the SV40 DTS on a plasmid has been
shown to increase nuclear import of the DNA in
nondividing cells by directly following the DNA using
fluorescently-labeled, triplex-forming peptide nucleic
acid (PNA) clamps and by in situ hybridization.7,44In
both cases, plasmids with the SV40 DTS localized to the
nuclei of nondividing or synchronized cells prior to cell
division, whereas plasmids lacking the sequence re-
mained in the cytoplasm. The DTS import activity has
also been followed in synchronized cells that were
microinjected with GFP-expressing plasmids by quanti-
fying gene expression.7Since the CMV immediate early
promoter does not mediate plasmid nuclear import, it
can be used to drive expression of a reporter gene in a
nuclear import assay. Matching CMV promoter driven
GFP plasmids containing or lacking the SV40 DTS
downstream of the GFP gene were microinjected into
either the cytoplasm or nuclei of synchronized cells.
Differences in GFP expression levels following nuclear
injection reflected differences in transcription, whereas
differences in expression following cytoplasmic injection
reflect DNA nuclear import activity. When equal
numbers of plasmids were injected into the nucleus,
they gave similar levels of GFP expression, suggesting
that the plasmids are transcriptionally
Further, injection of as few as 1–3 plasmids per nucleus
produced GFP+ cells at both 4 and 8 h postinjection.7
When as many as 1000 copies of the DTS-lacking plasmid
were injected into the cytoplasm of cells that divided at
14 h postinjection, no gene expression was detected prior
to cell division. By contrast, when anywhere between 10
and 1000 copies of the SV40 DTS-containing plasmid
were injected into the cytoplasm of these cells, gene
expression could be detected as early as 2–4 h following
microinjection. However, comparing the efficiency of
nuclear versus cytoplasmic injection, even with a DTS,
it took 20 times more DNA injected into the cytoplasm,
compared to the nucleus, to yield equivalent gene
Several other DNA sequences have been proposed to
act as DTSs. Mesika et al42cloned five repetitive NF-kB
binding sites into a luciferase-expressing plasmid and
found that gene expression was increased 12-fold
compared to the same plasmid lacking these binding
sites when transfected into cells. This increased gene
expression was enhanced by the addition of TNF-a, an
NF-kB activator. When the plasmids were fluorescently
labeled using rhodamine-labeled PNA clamps and
transfected into cells using dendrimers, the presence of
the NF-kB binding sites increased nuclear localization as
detected by confocal microscopy, and addition of TNF-a
increased nuclear localization even further. Thus, the
authors concluded that these NF-kB binding sites may
act to increase DNA nuclear import. Although many of
the described experiments used plasmids that also
Figure 2 Model for sequence-specific DNA nuclear import. Once in the cytoplasm, plasmids containing a DTS (depicted in yellow) can interact with newly
synthesized transcription factors to form protein–DNA complexes. Since transcription factors contain NLSs for their nuclear localization, the DTS-
containing DNA can become coated at distinct sites with one or more NLS, which can then interact with the importins to mediate nuclear import of the
entire complex. By contrast, plasmids lacking a DTS cannot form the appropriate DNA–protein complexes for nuclear import.
Nuclear entry of nonviral vectors
DA Dean et al
contained the SV40 early promoter and enhancer,
suggesting that the import detected could be due to a
combination of NF-kB and SV40 DTS activities, the
stimulation of import and expression by the NF-kB
binding sites suggests that these sequences do have DTS
activity. Indeed, more recent studies for the same group
have shown that NF-kB binding sites alone on a plasmid
can support nuclear import that is dependent on wild
type NF-kB p50 binding, confirming this model of DNA
Two other studies have developed matched DNA
binding site–transcription factor pairs that act as DTSs. In
one case, the OriP sequence from the Epstein–Barr virus
(EBV) was cloned into a plasmid and shown to increase
gene expression by six- to seven-fold following cyto-
plasmic microinjection in EBNA-1 expressing cells, when
corrected for transcriptional differences between the
plasmids and compared to plasmids lacking the OriP
sequence.45EBNA-1 is an EBV protein that binds to the
OriP sequence and contains an NLS. The authors
interpreted these experiments to suggest that the
EBNA-1 protein bound to the OriP containing plasmids
and aided nuclear import resulting in increased gene
expression. A more recent paper has used combinations
of the tet operator and a modified tetracycline repressor
containing an NLS (tetO and TetR-NLS, respectively) to
create a system in which DNA nuclear localization and
subsequent gene expression was increased.46When two
copies of the tetO were cloned into a plasmid and then
transfected into cells expressing TetR-NLS, gene expres-
sion increased almost 20-fold in growth-arrested cells,
and nuclear localization increased by four-fold. This
increased nuclear localization and expression was
dependent on both the presence of the tetO sequences
and the TetR-NLS protein. Further, the authors compared
the efficacy of the tet pair to plasmids bound to a PNA-
NLS conjugate and found that the PNA-NLS showed no
specific effects on nuclear localization or gene expres-
sion, suggesting that the spatial localization of the NLS
with respect to the DNA may be important for importin
interaction and nuclear import activity.
Cell-specific DNA nuclear import
Apart from the SV40 DTS and the other specific
examples just discussed, several DTSs have been
identified that act in cell-specific manners. Based on the
model proposed for DTS action, it was reasoned that
there may exist other DNA sequences that could act with
cell-specificity based on their ability to bind to cell-
specific transcription factors. Indeed, two such sequences
have been shown to have DNA nuclear import activity:
the smooth muscle gamma actin (SMGA) promoter and
the flk-1 promoter.32,47The SMGA gene is expressed in
smooth muscle cells and is regulated at the transcrip-
tional level by at least two factors, serum response factor
(SRF) and Nkx3.48–50When portions of this promoter
were placed into a pBR322-based plasmid and micro-
injected into the cytoplasm of smooth muscle cells, the
DNA localized to the nucleus in the absence of cell
division.32By contrast, when injected into other cell
types, such as endothelial cells, epithelial cells, or
fibroblasts, the plasmids remained in the cytoplasm.
Both SRF and Nkx3 play roles in the nuclear import of
the DNA. When SRF was expressed in CV1 cells, which
normally do not express this transcription factor and
support no nuclear import of the SMGA constructs,
detected.32Additionally, when binding sites for either
SRF or Nkx3 were mutated within the SMGA promoter,
nuclear import was abolished. This smooth muscle-
specific DTS also increased gene expression in trans-
fected growth-arrested smooth muscle cells, but not in
nondividing epithelial cells, suggesting that nuclear
import of plasmids can be restricted to specific cell types.
The flk-1 promoter also acts as a cell-specific DTS that
is functional only in endothelial cells. The flk-1 promoter
drives expression of a VEGF receptor and is restricted
largely to endothelial cells.51When several different
endothelial cell-specific promoters were cloned into the
CMV promoter-driven GFP nuclear import reporter
plasmid and tested for their ability to direct GFP
expression in nondividing cells by cytoplasmic micro-
injection, the flk-1 promoter showed nuclear import
activity, whereas three other promoters did not.47,52
Further, in situ hybridization confirmed that the flk-1
promoter caused nuclear import of the DNA in micro-
Unfortunately, there is no way to predict which
sequences will act as import sequences and which will
not. Thus, to identify DNA sequences with nuclear
import activity, a brute force approach has been taken in
which multiple cell-specific promoters are cloned into
appropriate vectors and tested for import activity by
microinjection or transfection assays. Hopefully as more
sequences are identified, common features will emerge,
allowing for a more directed approach for DTS identi-
Protein factor requirements for DNA nuclear
In order to better characterize the mechanisms of DNA
nuclear import, several groups have used digitonin-
permeabilized cells. This system has been extensively
utilized to characterize the mechanisms of signal-
mediated protein nuclear import and export.53Using
fluorescently labeled linear fragments of DNA, Hag-
strom demonstrated that nuclear import could be
detected in permeabilized cells and that import was
saturable and inhibited by agents that block the NPC or
deplete energy. However, import was not inhibitied by
excess NLS-containing proteins, suggesting that DNA
utilized a pathway distinct from that of NLS-containing
proteins. In support of this, it was found that these linear
DNA fragments were imported into the nuclei without
addition of exogenous cytoplasmic extracts, suggesting
that the importin machinery was not required. Further,
they demonstrated that DNA fragments larger than 1 kb
were excluded from the nuclei, while those smaller than
1 kb readily entered. This is in contrast to what has been
seen in intact cells, where plasmids up to 15 kb are
transported into the nucleus in the absence of cell
division.7,28,30However, in another study from the same
lab, when NLS peptides were fused to the DNA, larger
DNA fragments and even plasmids could be imported
into the nuclei of the permeabilized cells, but this time in
a manner that was absolutely dependent on cytoplasmic
Nuclear entry of nonviral vectors
DA Dean et al
extracts.18These results suggest that NLS-modified DNA
uses the NLS-dependent nuclear import machinery for
import whereas the NLS-free linear DNA did not.
Interestingly, one aspect to both of these studies that
may limit their relevance to the mechanisms used in
intact cells is the fact that in both cases, when the
substrates were microinjected into the cytoplasm of
intact cells, neither the fluorescently labeled linear
DNA fragments nor the NLS peptide-modified DNA
was able to localize to the nucleus.
Another study using the same permeabilized cell
system obtained different results that more closely
resembled the results found in intact cells.44In this
report, Wilson and co-workers used plasmids that were
fluorescently labeled at distinct sites with fluorescently
labeled PNA clamps.23,44When added to the permeabi-
lized cells, the DNA began to localize to the nuclei within
1 h and was abundantly imported by 4 h. As seen by
Wolff’s group, import was inhibited by WGA, energy-
depletion, and antibodies against the NPC. However,
unlike the Wolff group, import of intact plasmids
between 4 and 14 kbp was detected and required
cytoplasmic and nuclear extracts. The dependence of
cell extracts suggested that the importin machinery was
necessary for import. When recombinant importina,
importinb, and RAN were added to the DNA, no nuclear
import was observed, but when nuclear extracts were
also provided, DNA nuclear import was detected. Since
the importins cannot bind DNA directly, the nuclear
extracts likely provided transcription factors and other
DNA-binding proteins to act as adapters between the
importins and the DNA. Moreover, when a labeled
plasmid lacking the SV40 DTS was added to the
permeabilized cells in the presence of either cell extracts
or importina, b, RAN, and nuclear extracts, no nuclear
import was observed. These results support the model
for sequence-specific DNA nuclear import in which
transcription factors are needed to bridge the DTS
scaffold to the import machinery.
More recent experiments have studied the nuclear
import of several DNA-peptide or DNA-polymer con-
jugates and found that plasmids can indeed be imported
into the nuclei of permeabilized cells and that import is
energy-, time-, and NPC-dependent.13,54In one study, it
was even demonstrated that when an peptide containing
and RGD motif was linked to the DNAvia an oligolysine
peptide, nuclear import could be detected in permeabi-
lized cells leading to expression of a reporter gene.54
Finally, several other studies using isolated nuclei from
either cardiac myocytes or coconut syncitia demon-
strated that plasmids could be imported through the
NPC in a manner that was dependent on cytoplasmic
and nuclear extracts, as well as the presence of the SV40
DTS.55–57Moreover, these studies also demonstrated that
the isolated nuclei could not only import the DNA but
also export expressed mRNA leading to reporter gene
Approaches to increase DNA nuclear import
Most of the work aimed at characterizing the mechan-
isms of plasmid nuclear uptake has been done largely for
the purpose of trying to increase nuclear localization of
DNA to increase gene delivery and expression. To this
end, almost all of the focus on improving DNA nuclear
import has been on the use of NLS-containing peptides
or proteins as agents to drive the DNA into the nucleus
(Figure 3). Such proteins and peptides have been
complexed to DNA by electrostatic interactions,58–68
random covalent attachment to the DNA,18,69–72and by
site-specific attachment to the DNA using PNAs or
unique chemistries.73–79However, success has been
varied: some papers have reported that inclusion of
NLS-containing proteins or peptides increases gene
transfer and expression, while others have found no
such enhancement. A recent review by Cartier and
Reszka80has summarized the field beautifully and draws
a similar conclusion.
However, despite the lack of consensus on the positive
effects of inclusion of proteins or NLS-peptides to
increase nuclear trafficking and gene expression of
plasmids, several recent studies are worth mentioning,
if only for their novel approaches to nuclear targeting.
First, as discussed above, almost all attempts to increase
plasmid nuclear targeting have relied on using classical
NLS sequences. In contrast, one recent study has
covalently linked the importinb-binding (IBB) domain
from importina to plasmids and then transfected com-
plexes into cells.81During transport of classic NLS-
containing proteins, the NLS binds to importina, which
in turn binds to importinb through its IBB, and the NLS-
importina/b complex is transported into the nucleus.29
Scherman and co-workers ingeniously reasoned that by
binding the IBB to the DNA, only one intermolecular
interaction would need to form (eg, IBB-importinb) in
order for the complexed plasmid to be imported into the
nucleus. Unfortunately, although improved transfection
efficiency was observed using the IBB peptide, studies in
permeabilized cells indicated that the increased expres-
sion was not due to increased nuclear import, but rather
to the physicochemical properties of the IBB–DNA
Another unique approach to increase nuclear import
of plasmids has exploited the glucocorticoid receptor
(GR).82,83Under normal circumstances, GR resides in a
multiprotein complex in the cytoplasm with its NLS
masked. Upon ligand binding, the NLS is exposed and
the ligand-bound GR translocates into the nucleus.
Rebuffat et al attached the GR ligand dexamethasone to
plasmids by either a direct linkage via a psoralen linker
or using a PNA clamp. In both cases, the dexamethasone
reagents were able to interact with GR and induced
nuclear localization of the receptor. When steroid-
complexed DNA was transfected into nondividing cells
expressing GR, gene transfer and expression was 20- to
40-fold greater than that seen with unmodified DNA.
Thus, inclusion of a classic NLS-peptide is not the only
approach to improve nuclear trafficking and subsequent
In vivo DNA nuclear import
Despite the advances in characterizing the mechanisms
of DNA nuclear import in cultured and transfected cells,
there has been relatively little success transforming the
information gathered into better methods for DNA
delivery in vivo. Several papers have demonstrated that
complexation of DNA with NLS peptides can lead to
Nuclear entry of nonviral vectors
DA Dean et al
enhanced nuclear uptake and gene expression in zebra-
fish and shrimp embryos, but apart from these isolated
reports, most studies aimed at increasing DNA nuclear
import by addition of NLS-containing proteins or novel
polymers have remained restricted to cultured cell
studies.58,59,84The only successes in adult animals
reported to date have focused on the inclusion of DNA
nuclear targeting sequences to increase gene transfer and
expression in vivo. Incorporation of one or two copies of
the SV40 DTS into plasmids downstream of the reporter
gene resulted in a 20-fold increase in gene expression in
murine skeletal muscle following naked DNA injection
and electroporation.35,85When DNA was delivered
without electroporation, similar increases in gene ex-
pression were detected. Although the intracellular
localization of the DNA was not studied in these reports,
the authors proposed that the increases were due to the
nuclear import activity of the SV40 DTS and not classical
enhancer activity. More definitive proof for the in vivo
function of the SV40 DTS as a nuclear import sequence
came from studies by Young et al33on electroporation-
mediated gene transfer to the rat mesenteric vasculature.
In this study, inclusion of the SV40 DTS downstream of
CMV-driven reporter genes resulted in 40- to 200-fold
increased gene expression in intact arteries and veins.
When the location of the transferred DNA was deter-
mined by in situ hybridization, it was found that
plasmids lacking the SV40 sequence were present
throughout the tissue at 8 h but were cleared from the
tissue within 24 h (based on a lack of detectable signal),
but plasmids containing the SV40 DTS continued to be
detected at 24 and 48 h post-transfer. Moreover, plasmids
with an SV40 DTS localized to nuclei of cells in the tissue
even as early as 8 h and by 48 h, the only detectable
plasmid DNA was in the nuclei.
Unlike the situation seen in cultured cells where the
dependence on the DTS appeared almost absolute, a
number of studies have shown that in many tissues,
especially skeletal muscle, robust gene transfer and
expression can be obtained using plasmids lacking any
nuclear import sequence. Additionally, using highly
effective transfection reagents, certain cell types have
not shown an absolute dependence on the presence of a
DTS for gene transfer and expression. It is likely that
when the cytoplasm becomes filled with large concentra-
tions of plasmids, at least some of the plasmids can
randomly make their way to the nuclear envelope and be
imported into the nucleus independent of any DTS.
Indeed, when linear DNA is brought close enough to the
nuclear envelope using laser tweezers, it is pulled in.86
Further, it has been shown that when DTS-lacking
plasmids are delivered to the cytoplasm of a mouse
myotube in vivo, no gene expression is observed until
1 000 000 plasmids are injected, suggesting that mass
action could account for the DTS-independent nuclear
Figure 3 Various approaches to increase nuclear localization of transfected plasmids. A number of methods have been proposed and tested to increase the
nuclear import of plasmids following transfection. Most rely on the addition of NLS-peptides or NLS-containing proteins to the DNA, by either
electrostatic, covalent, or PNA clamps, to increase nuclear import. Other approaches have used general or cell-specific DTSs (in yellow), or synthetic DTS/
NLS-protein pairs (in blue).
Nuclear entry of nonviral vectors
DA Dean et al
It is clear that unless plasmids enter the nucleus, no gene
expression, integration, or replication of any vector DNA
can take place. Although the nuclear envelope breaks
down during mitosis thus eliminating this barrier to gene
transfer, most of the cells that are targets for gene therapy
are either slowly dividing or do not divide at all. Thus,
we must understand these mechanisms of plasmid
nuclear import if we are to develop ways to improve
intracellular trafficking. Despite the great focus on
development of better transfection reagents that increase
cell entry or the improvement of viral, nonviral, and
synthetic promoters to increase transcription, relatively
little success has been made at developing techniques
and reagents to increase nuclear DNA transport.
Hopefully, the elucidation of the basic mechanisms of
plasmid nuclear import will change this and lead to
more effective nonviral gene therapy approaches.
We thank Rui Zhou and Joshua Z Gasiorowski for
providing figures and all the members of our labs for
intriguing discussions and critical reading of the manu-
script. Work in the authors labs was supported in parts
by grants HL59956 (DAD), HL71643 (DAD), and
CA95608 (WEZ) from the NIH, and by The National
Medical Test Bed and the US Department of the Army
(Cooperative Agreement Number DAMD17-97-2-7016).
The content of the information does not necessarily
reflect the position or policy of the government or the
NMTB. DDS is supported by the Office of Research and
Development, Medical Research Service, Department of
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