(Received for publication, June 30, 1995; and in revised form, July 9, 1995)
From the
Systemic gene transfer provides new opportunities for the
analysis of gene function and gene regulation in vivo, as well
as for human gene therapy. We used the chloramphenicol
acetyltransferase reporter gene to examine several parameters important
for the development of efficient, cationic liposome-mediated,
intravenous (IV) gene transfer in mice. We then demonstrated that this
approach can produce high level expression of biologically important
genes. Specifically, we assessed the relationship of expression vector
design to the level of systemic gene expression produced, and compared
transfection levels produced by intravenously injecting DNA alone versus DNAliposome complexes. We found that both the
position of the heterologous intron, and the promoter element used in
the expression plasmid, significantly affected the level of systemic
gene expression produced. Although intravenous injection of plasmid DNA
alone transfected every tissue analyzed, liposome-mediated delivery was
much more efficient. We also established that repeated IV injection of
DNA
liposome complexes produced high level systemic transfection.
The second injection of DNA
liposome complexes produced levels of
gene expression at least as high as those following a single IV
injection. Thus, unlike some viral vectors, a neutralizing host-immune
response does not limit re-expression, following reinjection of
DNA
liposome complexes.
Finally, we showed that the expression vectors which produced the highest levels of chloramphenicol acetyltransferase reporter gene expression could also produce high level expression of two colony stimulating factor genes in mice. Specifically, IV injection of liposomes complexed to expression vectors into which we had inserted either the murine granulocyte-macrophage-colony stimulating factor cDNA or the human granulocyte-CSF cDNA, produced circulating levels of the corresponding colony stimulating factor gene product comparable to levels which have been shown previously to be both biologically and therapeutically significant.
The development of efficient, systemic transfer and expression
of cloned genes will permit analysis of gene regulation and gene
function, and the correction of a wide variety of genetic defects
directly in adults. Although systemic gene delivery has been reported
using both recombinant viral vectors and non-viral
vectors(1, 2, 3, 4, 5, 6, 7) ,
its utility is often limited by low level gene expression and/or
expression largely restricted to a single tissue. Cationic
liposome-mediated, IV gene delivery has been shown to produce
significant levels of reporter gene expression in all tissues
examined(7) . More recently, cationic liposome-mediated, IV
delivery of the wild type p53 gene has been shown to produce
significant anti-tumor effects in nude mice bearing human tumors
lacking p53 expression(8) . Transfer and expression of reporter
genes in mouse fetuses has also been demonstrated, following IV
injection of DNAliposome complexes into pregnant
mothers(9) . Furthermore, a single IV (
)injection of
a DNA expression plasmid alone containing the human tissue kallikrein
gene into spontaneously hypertensive rats produced a sustained
reduction in blood pressure for 6 weeks(10) .
To improve the
efficiency of liposome-mediated systemic gene transfer, we studied the
effects of the position of a heterologous intron in the expression
vector on the levels of CAT gene expression produced. We then used a
series of analogous expression plasmids, which differed only in the
promoter element, to directly compare the relative strengths of four
widely used viral promoter elements. Having established the vector
configuration that produced the highest levels of CAT gene expression,
we compared the transfection efficiency produced by IV injection of DNA
alone to injection of DNAliposome complexes. To assess whether we
could produce more persistent systemic gene expression, we analyzed the
level of CAT gene expression following reinjection of CAT expression
plasmid-liposome complexes. Previously, a neutralizing host-immune
response has been shown to limit re-expression, following reinjection
of some viral vectors into immunocompetent
animals(5, 6) . Finally, we showed that
liposome-mediated IV gene delivery can produce high level, systemic
expression of biologically and therapeutically relevant CSF genes.
A series of analogous plasmids,
pCMV-, pSV-
, pTK-
, and pAD-
, constructed by
MacGregor and Caskey(15) , each of which contain the SV40 late
gene 16 s/19 s splice donor/splice acceptor site, 5` to the
-galactosidase coding sequence, were purchased from Clontech. Each
was digested with NotI + ClaI, the blunt ends
filled in, and the vector fragment gel-purified. Then, the CAT gene in
a HindIII-BamHI fragment, derived from
pSV2-CAT(14) , was inserted by blunt end ligation, creating
plasmids pCMV-CAT, pSV-CAT, pTK-CAT, and pAD-CAT, respectively.
The coding sequence of mouse GM-CSF was amplified by PCR from a mGM-CSF-containing plasmid (kindly provided by Dr. A. Dunn, Ludwig Institute, Melbourne)(16) . The 5` PCR primer, 5`-GATCATCGATAGCGGCCGCCACCATGTGGCTGCAGAATTTACTTTTC-3`, contains a consensus initiation sequence(17) , and the coding sequence for the cDNA. The 3` PCR primer, 5`-GCTAGGTACCGCGGCCGCGATTCAGAGCTGGCCTGGGCTTCC-3`, contains 24 bases downstream of the TGA stop codon of the cDNA. The PCR fragment was digested with HindIII + KpnI, and the purified fragment ligated into the HindIII-KpnI site of p4109 (the vector containing the CMV promoter + 5` rat preproinsulin intron).
The coding sequence of the human G-CSF gene (kindly provided by Dr. R. Bosselman, Amgen) was digested with HindIII + SalI and the purified fragment was ligated into the HindIII-SalI site of p4136 (the vector containing the CMV promoter without a heterologous intron).
Effect of a heterologous intron on systemic gene transfer. Plasmids p4119, p4108, and p4121, all contain the CMV immediate early promoter-enhancer element linked to the CAT gene. P4108 lacks an intron, while p4119 contains a 375-bp intron sequence from the rat preproinsulin gene 5` to the CAT gene, and p4121 contains the same intron, 3` to the CAT gene. Groups of mice injected with either the 5` intron vector or the intronless vector showed significantly higher levels of CAT gene expression (p < 0.05) in every tissue analyzed than did mice injected with the 3` intron vector (Fig. 1). Tissues from the group of mice receiving the 5` intron plasmid showed 3- (liver) to 32- (skeletal muscle) fold higher levels of CAT activity, when compared to mice receiving the 3` intron plasmid p4121. Tissue CAT activity in mice receiving the 5` intron plasmid was not significantly higher than in mice receiving the intronless vector. Similar relative activities of p4119, p4108, and p4121 were also observed in in vitro transfections of CHO cells (Fig. 1). Northern analysis of mRNA isolated from these cells showed that the 5` and intronless vectors produced more appropriately spliced CAT mRNA than did the 3` intron vector (data not shown), in agreement with prior observations(23) . The ability of 5` heterologous introns to enhance gene expression has been attributed either to more efficient transcription (24) or alternatively to an increased accumulation of polyadenylated mRNA by a post-transcriptional mechanism(25) .
Figure 1:
CAT
activity in tissue extracts from mice which had received 960 nmol of
DDABcholesterol liposomes complexed to 60 µg of: p4119 (5`), p4108 (none), p4121 (3`), or a
CMV-luciferase plasmid (control). Mice were sacrificed 24 h
after receiving an IV tail vein injection of DNA
liposome
complexes in 200 µl of D5W. All values from each experiment include
four mice/group and represent mean ± standard deviation. #
indicates p < 0.05, when compared to either 3` or
control animals, as determined by a two-sided Student's t test. The plasmids were also transfected into CHO cells, as
described under ``Materials and
Methods.''
Figure 2: CAT activity in tissue extracts from mice which had received 960 nanomoles of DDAB:cholesterol liposomes complexed to 60 µg of p4119 (CMV-1)), pCMV-CAT (CMV-2)), pSV-CAT (SV40), pTK-CAT (TK), pAD-CAT (Adeno), or CMV-luciferase (control). Mice were sacrificed 24 h after receiving an IV tail vein injection. * indicates p < 0.0005, when compared to SV40, TK, adeno, or control animals, and + indicates p < 0.0005, when compared to control animals only.
Figure 3:
CAT activity in tissue extracts from mice
which had received 2 mg of p4119 alone (DNA alone) or 960 nmol
of DDAB/cholesterol (1:1) liposomes complexed to 60 µg of p4119 (DNAliposome complex) or CMV-luciferase (control). Mice were sacrificed 24 h after receiving an IV
tail vein injection of either naked DNA alone or DNA
liposome
complexes in 200 µl of D5W. * indicates p < 0.0005,
when compared to either DNA alone or control animals, and +
indicates p < 0.0005, when compared to control animals
only.
Animals sacrificed 21 days after a
single IV injection showed tissue CAT levels from 1% (liver) to 7%
(heart and skeletal muscle) of those in animals sacrificed 24 h after
injection (Fig. 4). (Peak tissue levels are present
approximately 24 h after a single injection of DNAliposome
complexes into mice(7, 22) .) Compared to control
animals, mice sacrificed 3 weeks after injection showed significantly
higher levels of CAT activity (p < 0.0005) in all tissues
assayed, except the liver. Thus, while still clearly detectable 21 days
after injection, CAT activity had fallen substantially from peak
levels. Mice sacrificed 24 h after their second injection showed levels
of CAT activity either as high or higher than levels in mice sacrificed
24 h after a single injection in every tissue assayed (Fig. 4).
Figure 4:
CAT
activity in tissue extracts from mice which had received a single IV
dose of p4119DDAB
cholesterol liposomes 24 h before
sacrifice (24 h) a single IV dose of
p4119
DDAB
cholesterol liposomes 3 weeks before sacrifice (3 wk), two IV doses of p4119
DDAB
cholesterol
liposomes, injected 3 weeks and then again 24 h before sacrifice (3
wk + 24 h), or a single IV dose of
CMV-luciferase
DDAB
cholesterol liposomes, 24 h before
sacrifice (control). * indicates p < 0.0005, when
compared to either 3 week or control animals. + indicates p < 0.0005, and # indicates p < 0.05, when
compared to control animals only.
The low levels of mGM-CSF observed following injection of
CAT plasmidDDAB
cholesterol liposome complexes suggests that
injection of DNA
liposome complexes may induce some release of
endogenous cytokines. However, despite this, IV injection of these CAT
plasmid
liposome complexes did not produce histopathologic changes
or any abnormalities in complete blood counts, serum chemistries, or
serum electrolyte values (data not shown).
Human G-CSF levels in serum ranged from 169 to 2,060 ng/ml in the hG-CSF gene-treated mice, but were not detectable in serum from any of the CAT gene-treated mice. The wide range of hG-CSF activities seen in the sera from individual mice treated with the hG-CSF gene contrasted with the narrow range of hG-CSF levels in the lung, assayed in the same group of animals (Table 2). This suggests that variable serum levels reflected variability in serum pharmacokinetics in these animals, rather than differences in the efficiency of the intravenous injection itself between the individual animals.
We sought to improve expression vector design for intravenous gene delivery by varying several components of the vector. The most efficient expression vectors we constructed produced high level expression not only of the CAT reporter gene, but also of several biologically important colony stimulating factor genes. Specifically, we investigated the effects of different viral promoters and intron sequences within the expression vector on the efficiency of liposome-mediated, systemic gene expression. We found that the effects of such sequences on expression of the genes transferred intravenously may differ from results obtained previously either in transgenic animals or by non-systemic routes of in vivo gene delivery. For example, prior studies in transgenic animals have shown that cDNAs are expressed at significantly higher levels when the transgene incorporates a heterologous intron 5` to the coding region. Analogous transgenes, either lacking an intron or containing a 3` intron are expressed at comparatively lower levels(23, 24, 25, 26) . While we have also found that liposome-based, IV injection of a vector with a 5` intron produced significantly higher levels of gene expression than the analogous 3` construct, it did not result in significantly higher levels than the vector lacking an intron (Fig. 1). Prior studies demonstrating the importance of 5` heterologous intron sequences in producing high level expression of plasmid cDNA constructs have analyzed the expression of integrated transgenes in germline transgenic mice(24, 26) . In contrast, the genes transferred by liposome-mediated IV injection remain largely episomal(7, 20) . Moreover, genes injected IV into adult animals are not exposed to the developmental influences to which transgenes integrated into mouse chromatin are exposed(24) .
Our survey of four commonly used viral promoter elements demonstrated that the CMV promoter is more active than adenovirus, SV40, and TK promoters in all tissues analyzed, following systemic delivery (Fig. 2). Previously, adenoviral, SV40, and CMV promoters have been shown to produce comparable levels of gene expression in a number of these tissues, following localized gene delivery by particle bombardment in rats(27) . The differences in viral promoter activity we observed may relate to the mode of gene transfer used or to species variability. They may also reflect differences in the predominant cellular sites of gene expression produced by systemic gene delivery. Our prior immunohistochemical analyses have indicated that IV-injected genes are expressed in large numbers of cells located primarily within the vascular compartment (7, and data not shown). This compartment appears relatively inaccessible to genes administered extravascularly(27) .
Using p4119, one
of our most efficient CMV-based expression vectors, we found that IV
injection of naked p4119 plasmid transfected every tissue. In agreement
with this observation, IV injection of a human tissue kallikrein gene
expression plasmid alone into hypertensive rats has recently been
reported to produce significant reductions in blood
pressure(10) . However, we found that cationic
liposome-mediated IV delivery of our most efficient expression plasmid
increased the efficiency of in vivo gene expression over
vector alone by up to 3 orders of magnitude (Fig. 3). We have
also found that the modes by which naked DNA and DNAliposome
complexes transfect cells, following IV injection into mice appear to
differ. Intravenous preinjection of cationic liposomes, 20 min prior to
injecting p4119-liposome complexes, significantly reduced (p < 0.005) CAT gene expression in all tissues, whereas
preinjecting cationic liposomes 20 min prior to injecting p4119 alone
either had no effect, or in some tissues, increased CAT gene expression
(data not shown). Thus, preinjection of liposomes appears to block
uptake or expression of DNA
liposome complexes, but not of plasmid
DNA alone.
Prior studies using systemic injection of recombinant
adenoviral vectors have shown that a neutralizing host immune response
limits re-expression of the transferred gene, following reinjection of
the adenoviral vector(5, 6) . In contrast, we found
that reinjecting DNAliposome complexes into immunocompetent mice
3 weeks after an initial IV injection produced peak levels of gene
expression at least as high as those following a single IV injection (Fig. 4). Thus, DNA
liposome complexes can be reinjected at
least one time, without any apparent reduction in the efficiency of
gene transfer and expression.
Finally, using two optimized,
CMV-based expression vectors, we assessed IV, cationic
liposome-mediated transfer and expression of the murine GM-CSF and the
human G-CSF genes. Intravenous injection of liposomes complexed to our
CMV-based vector containing a 5` rat preproinsulin intron and the
murine GM-CSF cDNA produced mean serum GM-CSF protein levels
approximately 240 pg/ml above control levels 24 h later (Table 1). In contrast, studies by others (28) have shown
that serum mGM-CSF levels become undetectable (<10 pg/ml) by 24 h
after subcutaneous injection of 100 ng of recombinant murine GM-CSF
protein into mice. Furthermore, the serum mGM-CSF levels we produced by
injecting mGM-CSF gene liposome complexes appear comparable to those
produced in mice 24 h after sub-cutaneous injection of 10 fibrosarcoma cells, stably transfected with the mGM-CSF gene, and
secreting high levels of mGM-CSF protein(28) . Either a single
injection of 10
fibrosacroma cells transfected with mGM-CSF
or multiple subcutaneous injections of 100 ng of mGM-CSF protein
substantially elevated white blood cell counts in
cyclophosphamide-treated mice(28) . Since we appear to have
produced comparable serum levels of mGM-CSF, these are likely to be
biologically and therapeutically significant.
We also injected
hG-CSF geneliposome complexes into mice. Administration of
recombinant human G-CSF protein has been shown to significantly
accelerate the recovery of white blood cell counts, following either
the administration of cytotoxic agents or bone marrow
transplantation(29, 30) . Recombinant human G-CSF is
now approved by the FDA for treatment of myelosuppression in human
patients (30) and is thus relevant for use as a potential gene
therapy. In addition, human G-CSF protein does not cross-react with
murine G-CSF in G-CSF ELISA assays. (
)Therefore, unlike
expression of our mGM-CSF vector, human G-CSF activity can be assayed
in mice with essentially no background activity. Intravenous injection
of cationic liposomes complexed to our CMV-based vector containing the
human G-CSF cDNA but lacking a heterologous intron produced mean serum
hG-CSF protein levels approximately 1,000 pg/ml above control levels 24
h later. These levels are approximately 100 times higher than serum
G-CSF levels normally present in humans. (G-CSF levels in normal humans
are routinely at or below the limits of detection by
ELISA(31) .) Furthermore, the serum G-CSF levels we observed in
hG-CSF gene-treated mice are comparable to serum G-CSF levels present
in human patients with either acute infectious diseases (32, 33) or following myeloablative chemotherapy and
subsequent bone marrow transplantation (34) . Both of these
conditions are associated with marked elevations of serum G-CSF
levels(32, 33, 34) . Thus, IV liposome-based
delivery of the human G-CSF gene appears to produce levels of G-CSF
gene expression which may produce biologic and therapeutic effects.
In conclusion, we have explored several parameters that affect the efficiency of systemic gene transfer and expression. Our results show that the design of the expression plasmid is critical in determining the level of gene expression achieved. The CAT reporter gene, whose expression can be sensitively and specifically measured in mammalian cells(14) , has been useful in developing in vivo gene delivery strategies that permit high level expression of biologically important genes. Further improvements in the design of both expression vectors and DNA carriers for systemic gene transfer may produce more efficient expression of transferred genes and ultimately permit more effective treatment of a wide variety of genetic diseases.