©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cationic Liposome-mediated Intravenous Gene Delivery (*)

(Received for publication, June 30, 1995; and in revised form, July 9, 1995)

Yong Liu (1) Denny Liggitt (2) Wendy Zhong (1) Guanhuan Tu (1) Karin Gaensler (3) Robert Debs (1) (3)(§)

From the  (1)California Pacific Medical Research Institute, San Francisco, California 94115, the (2)Department of Comparative Medicine, University of Washington, Seattle, Washington 98195, and the (3)Department of Medicine, University of California, San Francisco, San Francisco, California 94143

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 DNAbulletliposome 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 DNAbulletliposome complexes produced high level systemic transfection. The second injection of DNAbulletliposome 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 DNAbulletliposome 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.


INTRODUCTION

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 DNAbulletliposome complexes into pregnant mothers(9) . Furthermore, a single IV (^1)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 DNAbulletliposome 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.


MATERIALS AND METHODS

Animals

Two-month-old, female, ICR mice obtained from Simonsen Laboratories, Gilroy, CA were used in all experiments.

Plasmid Construction

Details of the plasmid constructions are provided(11) . In brief, plasmids p4108, p4119, and p4121 each contain a composite human cytomegalovirus (CMV) promoter derived from the Towne strain, 5` to the internal NcoI site of the enhancer (from pCATwt760)(12) , and 3` of the NcoI site from the AD169 strain (from pBC12/CMV/IL2)(13) , and the CAT coding sequence from pSV2-CAT(14) . Plasmid p4108 has no intron. Plasmid p4119 includes an intron (a 375-bp fragment from the rat preproinsulin gene from pBC12/CMV/IL2(13) ), 5` to the CAT gene. Plasmid p4121 is similar to p4119, except that the intron was ligated 3` of the CAT gene.

A series of analogous plasmids, pCMV-beta, pSV-beta, pTK-beta, and pAD-beta, 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 beta-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).

Preparation of Plasmid DNA

Plasmids were purified using alkaline lysis and ammonium acetate precipitation with PEG extraction(18) , and the nucleic acid concentration measured by UV absorption at 260 nm. The concentration of each plasmid was verified on agarose gels to ensure that equal amounts of the different plasmids were injected per mouse.

Preparation of Cationic Liposomes

Liposomes containing the cationic lipid dimethyldioctadecyl ammonium bromide (DDAB) in a 1:1 molar ratio with cholesterol were prepared as described previously(19) , except that the dried lipid film was resuspended in 5% dextrose in water (D5W), and then sonicated in a bath sonicator for 20 min. DDAB was purchased from Sigma and cholesterol from Calbiochem. The liposomes were stored under argon at 4 °C until use. Prior to mixing, the liposomes and the plasmid DNA were allowed to equilibrate to room temperature for 20 min.

Transfection of Chinese Hamster Ovary (CHO) Cells

CHO cells, obtained from the UCSF cell culture facility, were maintained in RPMI 1640 with 10% fetal bovine serum at 37 °C in 5% CO(2). Two sets of triplicate plates were transfected with p4119, p4108, or p4121, as described previously(20) . One set of plates was harvested 24 h later, mRNA isolated, and probed for CAT mRNA. The other set of plates were harvested at 48 h and extracts analyzed for CAT activity (below).

Preparation of DNAbulletLiposome Complexes for IV Injection of Mice

Mice were injected in groups of four. For each group of animals, 360 µg of plasmid DNA in 600 µl of D5W was rapidly pipetted into a sterile, 1.7-ml Eppendorf tube containing DDAB-cholesterol liposomes (5.760 µmol of total lipid) in 600 µl of D5W. The mixture was then pipetted up and down two times and was not agitated further. (The mixture turned cloudy, but no precipitates or aggregation were visible.) Each animal then received 200 µl of the DNAbulletliposome complexes in D5W, containing 60 µg of plasmid DNA (2 mg/kg) complexed to 960 nmol of DDAB-cholesterol liposomes, by tail vein injection, using a 1-ml syringe and 27-gauge needle. The control group received 60 µg of a CMV-luciferase plasmid, complexed to 960 nmol of DDAB-cholesterol liposomes. (Levels of CAT gene expression in tissues from animals treated with the CMV-luciferase plasmid-liposome complexes did not differ significantly from those in untreated mice (data not shown).) Mice receiving p4119 alone received 2 mg in 200 µl of D5W by tail vein. Each experiment was repeated as least once and yielded comparable results. All mice appeared normal from the time of injection until sacrifice in every experiment.

Assay of CAT Activity

Multiple tissues were dissected from animals sacrificed either 1 or 21 days following IV injection and processed as described(21) . Protein concentrations were normalized for each set of tissues assayed and a volume of each extract was added to 10 µl of a 20 mM acetyl CoA stock solution in water (Sigma), containing 0.3 µCi of ^14C-labeled chloramphenicol (Amersham Corp.). Homogenization buffer was added to adjust final sample volume to 122 µl, and the mixture was allowed to react at 37 °C for 8 h. CAT activity was then measured (14) and CAT units determined (22) as described previously.

Assay of Murine GM-CSF Levels

The ELISA for the detection of mGM-CSF was established using a monoclonal rat anti-mouse GM-CSF antibody (Pharmingen, San Diego, CA.) as the capture antibody. A biotinylated monoclonal rat anti-mouse GM-CSF antibody was used as the secondary antibody (Pharmingen). Standard values were generated using a recombinant mouse GM-CSF calibrated against a commercially available standard (Endogen, Boston, MA). The conjugate was peroxidase-Z-avidin (Z-HRP, Zymed, So. San Francisco, CA.) and the substrate was ABTS-soluble substrate kit (Zymed). The procedure for detection of GM-CSF by the ELISA is as follows.

Plate Preparation

The ELISA plates (Immulon 2 flat bottom microtiter plate, Dynatech Laboratories, Chantilly, VA) were prepared 24 h in advance, by coating each well with 50 µl of a 1:100 dilution of monoclonal rat anti-mouse GM-CSF antibody in a 50 mM sodium bicarbonate buffer (pH 9.4). The plates were then treated with a blocking solution containing 2% BSA (98-99% Albumin, Sigma) in 100 mM NaCl, 10 mM Na(2)HPO(4), 2 mM NaCl, 2 mM KH2PO4 in PBS added to each well. The plates were incubated for 2 h at room temperature, and then washed five times with PBS containing 0.2% Tween-20 (PBST).

Assay Procedure

The standard was diluted in 2% BSA in PBS to establish a range from 1 to 0.025 ng/ml. One-hundred µl of each dilution of the standard was added in triplicate. Blood was aspirated directly from the right ventricle with a 23-guage needle, transferred to capillary serum separator tubes (Sherwood, St. Louis, MO), incubated for 1 h at 4 °C, and then centrifuged at 10,000 revolutions/min for 10 min in an Eppendorf microcentrifuge to recover the serum. The lung tissue samples were homogenized in 1 µl of PBS and centrifuged at 10,000 revolutions/min for 10 min. One-hundred µl of supernatant from each sample was added in triplicate and the plates incubated for 12 h at 4 °C. Plates were washed five times with PBST. One-hundred µl of a 1:1000 dilution of biotinylated rat anti-mouse GM-CSF antibody in 2% BSA in PBS was added to each well and the plates incubated for 1 h at room temperature. The plates were washed five times with PBST. One-hundred µl of a 1:500 dilution of Z-HRP in 2% BSA in PBS was added to each well and the plates incubated for 1 h at room temperature. The plates were then washed five times with PBST, and 100 µl of the ABTS substrate added to each well. Following incubation for 1 h, the plates were read at 405 nm using a Multiskan MKII micro plate reader (Flow Titertek, Mclean, VA.) The results were analyzed using Immunofit EUA/RIA Analysis software (Beckman, Palo Alto, CA).

Assay of Human G-CSF Levels

Human granulocyte colony stimulating factor (hG-CSF) was detected using an ELISA kit (R& Systems, Minneapolis, MN). Samples were prepared and results analyzed in a similar fashion as for the GM-CSF assay except that dilutions were performed as directed by the manufacturer.


RESULTS

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 DDABbulletcholesterol 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 DNAbulletliposome 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.''



In Vivo Comparison of Four Different Viral Promoter Elements

We compared the relative strengths of the CMV immediate early promoter, the SV40 early promoter, the herpes simplex virus thymidine kinase (TK) promoter, and the adenovirus 2 major late promoter fused to the adenovirus tripartite leader. To directly compare them, we used a series of CAT expression plasmids constructed identically except for the promoter element(12) . Each of these plasmids contains an intron (derived from the SV40 late gene) 5` to the coding sequence. We also tested p4119, which contains an alternative 5` intron (derived from the preproinsulin gene) and the CMV promoter. The two CMV-CAT plasmids produced similar levels of tissue CAT gene expression, regardless of the intron used. Both of these plasmids produced much higher levels of CAT gene expression (p < 0.0005) in every tissue assayed than did the SV40-, TK- or adenoviral-CAT plasmids (Fig. 2). The CMV-CAT expression plasmid produced 10-55-fold higher levels of CAT activity than did the analogous SV4O-CAT plasmid, 24-273-fold higher levels than did the TK-CAT plasmid, and 14-46-fold higher levels than the adenoviral-CAT plasmid, depending on the tissue.


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.



Comparison of IV Delivery of DNA Alone Versus DNAbulletLiposome Complexes

We assessed whether IV injection of a large dose of our most efficient expression plasmid, p4119 alone, could produce systemic transfection, and compared its efficiency to that produced by injection of p4119-liposome complexes. Mice received either 2 mg (66 mg/kg) of p4119 alone or 60 µg (2 mg/kg) of p4119 complexed to cationic liposomes, as a single IV injection. Mice receiving the CMV-CAT plasmid DNA alone showed significantly higher levels of CAT activity than controls (p < 0.0005) in every tissue (Fig. 3). However, p4119-liposome complexes produced much more efficient in vivo transfection (p < 0.0005) than did p4119 alone. Mice receiving DNAbulletliposome complexes showed from 57-fold higher tissue CAT levels in the heart to 1000-fold higher levels in the spleen per µg of DNA injected. IV injection of p4119 doses lower than 2 mg produced proportionately lower amounts of tissue CAT activity (data not shown).


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 (DNAbulletliposome complex) or CMV-luciferase (control). Mice were sacrificed 24 h after receiving an IV tail vein injection of either naked DNA alone or DNAbulletliposome 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.



Gene Expression Following Reinjection of DNAbulletLiposome Complexes

We assessed whether reinjection of p4119-liposome complexes could produce high level re-expression of the CAT gene. Initially, two groups of four mice received p4119bulletDDABbulletcholesterol complexes injected IV. Three weeks later, one group was sacrificed. At that time, the other group was reinjected with a second, identical dose of the p4119-liposome complexes. Also, at this time, groups of four previously uninjected littermate mice received an identical IV injection of p4119-liposome complexes or CMV-luciferase-liposome complexes (control mice). Twenty-four h later, the remaining three groups were sacrificed and tissue CAT activities measured.

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 DNAbulletliposome 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 p4119bulletDDABbulletcholesterol liposomes 24 h before sacrifice (24 h) a single IV dose of p4119bulletDDABbulletcholesterol liposomes 3 weeks before sacrifice (3 wk), two IV doses of p4119bulletDDABbulletcholesterol liposomes, injected 3 weeks and then again 24 h before sacrifice (3 wk + 24 h), or a single IV dose of CMV-luciferasebulletDDABbulletcholesterol 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.



Circulating Levels of Murine GM-CSF Protein following IV Injection of mGM-CSF Gene Expression Plasmid-Liposome Complexes.

We then assessed whether the expression plasmidcationic liposome complexes that produced the highest levels of CAT gene expression ( Fig. 1and Fig. 2) could also produce high level expression of an important cytokine gene. Specifically, we tested whether IV, cationic liposome-mediated delivery of the murine GM-CSF cDNA, ligated into our CMV-based expression vector containing the 5` rat preproinsulin intron (Fig. 1) could produce biologically and therapeutically significant circulating levels of murine GM-CSF protein in mice. We measured serum GM-CSF levels 24 h following IV injection of DDABbulletcholesterol liposomes complexed to this expression vector containing either the murine GM-CSF cDNA or the CAT cDNA (p4119). The group of mice injected with the GM-CSF genebulletliposome complexes had mean serum GM-CSF levels of 332 ± 110 pg/ml, whereas mice injected with CAT genebulletliposome complexes had mGM-CSF levels of 93 ± 27 pg/ml (p < 0.005 by Student's t test) (Table 1). Thus, injection of the GM-CSF expression plasmidbulletliposome complexes produced significant circulating levels of GM-CSF protein 24 h after injection.



The low levels of mGM-CSF observed following injection of CAT plasmidbulletDDABbulletcholesterol liposome complexes suggests that injection of DNAbulletliposome complexes may induce some release of endogenous cytokines. However, despite this, IV injection of these CAT plasmidbulletliposome complexes did not produce histopathologic changes or any abnormalities in complete blood counts, serum chemistries, or serum electrolyte values (data not shown).

Circulating and Tissue Levels of Human G-CSF Protein Following IV Injection of G-CSF Expression PlasmidbulletLiposome Complexes

Finally, we assessed IV, cationic liposome-mediated transfer and expression of the human G-CSF gene in mice. The human G-CSF cDNA was ligated into our CMV-based based expression vector lacking a heterologous intron (Fig. 1). Subsequently, we measured human G-CSF levels in serum, as well as G-CSF levels in the lung tissue, 24 h following IV injection of DDABbulletcholesterol liposomes complexed to this expression vector containing either the human G-CSF cDNA or the CAT cDNA (p4108). The group of mice injected with the G-CSF genebulletliposome complexes had mean lung G-CSF levels of 141 ± 10 pg/mg of tissue and serum levels of 1,012 ± 710 pg/ml of serum, whereas mice injected with CAT genebulletliposome complexes had undetectable human G-CSF levels (<10 pg/ml) in either lung or serum (p < 0.005 by Student's t test for both lung and serum) (Table 2). Thus, IV injection of the G-CSF expression plasmidbulletliposome complexes into mice produced significant circulating and tissue levels of human G-CSF protein 24 h later.



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.


DISCUSSION

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 DNAbulletliposome 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 DNAbulletliposome 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 DNAbulletliposome 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, DNAbulletliposome 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 10^7 fibrosarcoma cells, stably transfected with the mGM-CSF gene, and secreting high levels of mGM-CSF protein(28) . Either a single injection of 10^7 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 genebulletliposome 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. (^2)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.


FOOTNOTES

*
This work was supported by National Institutes of Health R01 Grants DK45917, CA58914, and HL53762 (to R. J. D.), and in part by Megabios Corp, Burlingame, CA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed.

(^1)
The abbreviations used are: IV, intravenous; CAT, chloramphenicol acetyltransferase; CSF, colony stimulating factor; CMV, cytomegalovirus; bp, base pair(s); GM-CSF, granulocyte-macrophage-CSF; PCR, polymerase chain reaction; m, murine; DDAB, dimethyldioctadecyl ammonium bromide; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ABTS, 2,2-azino-di-(3-ethylbenzthiazoline sulfonic acid).

(^2)
R and D Systems, personal communication (technical information).


ACKNOWLEDGEMENTS

We thank Drs. Tim Heath and John McLean for their assistance.


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