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Evaluation of Cationic Liposome Suitable for Gene Transfer into Pregnant Animals
Biochemical and Biophysical Research Communications 258, 358 –365 (1999) Article ID bbrc.1999.0590, available online at http://www.idealibrary.com on
Evaluation of Cationic Liposome Suitable for Gene Transfer into Pregnant Animals Takahiro Ochiya,* Yasushi Takahama,* Hiroyasu Baba-Toriyama,† Makoto Tsukamoto,‡ Yuko Yasuda,§ Hiroshi Kikuchi, ¶ and Masaaki Terada* ,1 *Genetics Division and †Chemotherapy Division, National Cancer Center Research Institute, Tokyo, Japan; ‡Internal Medicine, Chiba University School of Medicine, Chiba, Japan; §Louis Pasteur Center for Medical Research, Kyoto, Japan; and ¶Daiichi Seiyaku Co., Ltd. Research Institute, Tokyo, Japan
Received March 25, 1999
Cationic liposome-mediated in vivo gene transfer represents a promising approach for somatic gene therapy. To assess the most suitable liposome for gene delivery into a wide range of organs and fetuses in mice, we have explored several types of cationic liposomes conjugated with plasmid DNA carrying the b-galactosidase gene through intravenous injection into pregnant animals. Transduction efficiency was assessed by Southern blot analysis and expression of the transferred gene was evaluated by enzymatic demonstration of b-galactosidase activity. Through the analysis of several types of recently synthesized cationic liposome/lipid formulations, DMRIE-C reagent, a liposome formulation of the cationic lipid DMRIE (1,2dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) and cholesterol in membrane-filtered water met our requirements. When the plasmid DNA/ DMRIE-C complexes were administered intravenously into pregnant mice at day 11.5 post coitus (p.c.), transferred genes were observed in several organs in dams and were expressed. Furthermore, although the copy numbers transferred into embryos were low, we observed reporter gene expression in the progeny. © 1999 Academic Press
Key Words: cationic liposome; gene therapy; fetus.
Recently, nucleic acids are delivered to cells by transduction with viral particles or by transfection which includes chemical or physical methods. Cationic liposomes have been used mostly in in vitro gene transfer experiments because they offer several advantages over other chemical means, such as calcium phosphate coprecipitation or DEAE-Dextran (1, 2, 3). These advantages include: (1) non-viral delivery system mini1 To whom correspondence should be addressed. National Cancer Center Research Institute 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo, 104 Japan. Fax: 81-3-3541-2685. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
mizing concerns related to public health and vector safety; (2) availability to produce transient and stable transfection in vitro; (3) ability to transfer a variety of macromolecules, including several types of DNA with plasmid, oligonucleotide and yeast artificial chromosome, RNA and protein; (4) ease of large quantity liposome production; (5) repeatable administration in vivo; (6) protection of plasmid DNA molecules from degradation by nucleases and shearing by aerosolisation; (7) relatively low cytotoxicity for mammalian cells. Several features of these nonviral vectors are attractive for gene transfer studies in vivo, including their relative safety and lack of toxicity (4-11). However, a limitation of these cationic liposomes has been a relative inefficiency of gene transfer in vivo. We previously reported that a single intravenous (i.v.) injection of foreign gene-cationic lipopolyamine containing dioctadecylamido-glycylspermine (DOGS) (12) complexes into pregnant mice enables transgenes to be expressed in the fetuses and postpartum progeny (13). However, we and others later noticed that the efficiency of the gene transfer into fetuses is varied and the batches of DOGS currently available allowed no positive results. We do not know the reason why we have had difficulty in reproducing with consistent efficiency a gene transfer by our previous method by using different batches of DOGS. But, we have observed that the DNA/DOGS complex often made a large number of aggregates when current batches of DOGS were used. The aggregates may be trapped in the lung, which in turn produced an unsuccessful gene transfer into the fetus. The extent of the aggregates may depend on the purity of plasmid DNA, incubation time, temperature, and the liposome batch and differ from experiment to experiment. Studies were performed to determine optimal cationic liposome formulation for successful gene transfer into adult animals or progeny of the pregnant animals. The safety and toxicity of those lipid
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FIG. 1. Systemic gene delivery of plasmid DNA to pregnant animals. The pCMV z SPORT-bgal expression plasmid was used to assess the gene transfer efficiency of a number of different cationic liposomes in vivo. Liposome/DNA complex was administered into pregnant animals at day 11.5 p.c. Animals were sacrificed two days after the administration and then the genomic DNAs were extracted from embryos and maternal liver and subjected to Southern blot analysis. Among 8 cationic liposomes that were tested, DMRIE-C reagent allowed maximum transduction of the genes into maternal liver (Fig. 1a) as well as fetus (Fig. 1b). Each lane contained 10 mg of undigested genomic DNA. As a positive control, 100 pg of uncut (supercoiled) and linealized plasmid DNA were loaded.
formulations were also assessed in vivo after systemic i.v. injection into mice. Here, we show that the DMRIE-C reagent allows a reproducible and an efficient systemic delivery of genes into several organs of dams and allows expression of the transferred genes. In their progeny, although in a small amount, we observed gene transduction and expression. MATERIALS AND METHODS Plasmid expression vector and cationic lipids. An eukaryotic expression vector plasmid containing b-galactosidase gene, pCMV z SPORT-bgal (Life Technologies Inc.), was used for all the experiments. The plasmid DNA was purified through Wizard Plasmid Purification System (Promega), ethanol-precipitated twice, and finally dissolved in high quality, nuclease-free water. Cationic lipid was purchased: Transfectam (12) (DOGS: dioctadecyldimethylammonium chloride) and Tfx-50 (14) (cationic lipid [N,N,N9,N9-tetramethyl-N,N9-bis(2-hydroxy-ethyl)-2,3,-dioleoyloxy1,4-butanediammonium iodide]/DOPE: dioleoylphosphatidylethanolamine) from Promega, USA; Lipofectin (15) (DOTMA: N-(2,3(dioleoyloxy)propyl)-N,N,N-trimethyl ammonium chloride/DOPE: dioleoylphosphatidylethanolamine), LipofectAMINE (16) (DOSPA: 2,3-dioeoyloxy-N-(2(spermidinecarboxamodo)-ethyl)-N,N-dimethyl-
1-propanaminium trifluoroacetate/DOPE), LipofectAce (17) (DDAB: dimethyldioctadecylammonium bromide/DOPE) and DMRIE-C (18) (DMRIE: 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide/cholesterol) from Life Technologies Inc.; Gene Transfer (19) (TMAG: a-(trimethylammonioacetyl)-didodecyl D-glutamete chloride/DLPC: dilauroyl phosphatidylcholine/DOPE) from Wako, Japan; ExGen 500 (20) (cationic polymer polyethylenimine) from Euromedex. To determine the optimal ratio of cationic liposome to plasmid DNA, pCMV z SPORT-bgal was complexed with indicated liposomes at increasing amounts of 10 mg to 1000 mg whereas the DNA was held constant at 100 mg. Other non-commercialized liposomes including different molar ratios of DOTMA/DOPE, TMAG/ DLPC/DOPE were generously gifted from Daiichi Seiyaku Co. Ltd. Research Institute. In total, 26 cationic liposomes were tested. In vivo gene transfer. Male and female adult mice (7-week old) and pregnant ICR mice at day 11.5 p.c. were used for in vivo experiments. The DNA/liposome complex for in vivo administration was prepared as follows. A 250 ml solution of cationic liposome containing 10-1000 mg of DMRIE-C reagent was added to 250 ml of PBS(2) containing 100 mg of plasmid DNA drop by drop (not the reverse order), then mixed gently by drawing and withdrawing the solution into and out of the syringe three to five times. A cloudy solution may appear. The mixed solution was incubated at room temperature for 6 to 7 min. Incubation for more than 8 min leads to excess aggregates which may result in a less efficient transfer
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of the gene. Just before administration into animals, the solution was mixed by inverting the tube 2-3 times. The mixed solution should not have visible aggregates. The entire solution was then drawn into a disposable 1 ml plastic syringe fitted with a 27G 33/4 needle, and injected into the tail vein of adult male and female animals and pregnant ICR mice at day 11.5. The animals were lightly anesthetized when given intravenous injections, and the injection speed was about 0.5 ml/4-5 seconds. Concerning other liposomes tested in this experiment, the DNA and lipid were each diluted to 0.25 ml with PBS(2) and then mixed by adding the liposome solution to the DNA drop by drop. The optimal ratios of DNA to liposomes (wt/wt) are: DOGS, 1:5; Tfx-50, 1:5; Lipofectin, 1:10; LipofectAMINE, 1:5; LipofectAce, 1:10; Gene Transfer, 1:5; ExGen 500, 1:10; DOTMA/DOPE, 1:5; TMAG/DLPC/DOPE, 1:5, which were determined by in vitro transfection analysis by the use of primary cultures from fetal mice fibroblasts (unpublished observations). The DNA/liposome complexes from Lipofectin, LipofectAMINE and LipofectAce were administered immediately into mice. The other complexes from DOGS, Tfx-50, Gene Transfer, DOTMA/DOPE, TMAG/DLPC/ DOPE and ExGen 500 were incubated 5 to 10 min at room temperature and then used. Southern analysis for the detection of the transgene. The genomic DNA from whole embryo bodies and their maternal organs was subjected to Southern blot analysis for the presence of the b-galactosidase gene. Ten mg of genomic DNA was separated by electrophoresis on a 0.8% agarose gel, transferred onto a nylon membrane (Hybond N plus, Amersham), and hybridized with a a 32 P-labeled b-galactosidase fragment DNA as a probe in 5 3 Denhardt’s solution (1 3 Denhardt’s 5 0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 0.5% SDS, 5 3 sodium saline phosphate EDTA (1 3 sodium saline phosphate EDTA 5 150 mM NaCl, 10 mM sodium phosphate, 1.3 mM EDTA), and 200 mg/ml of salmon testis DNA at 65°C for 16 hr. The filters were then washed in 0.1% SDS and 0.1 3 sodium saline phosphate EDTA at 65°C. Assay of b-galactosidase activity in vitro. b-galactosidase activity was measured with b-Galactosidase Enzyme Assay System (Promega). In brief, homogenized tissues were washed with PBS(2) several times, and 1 ml of 1x Reporter Lysis Buffer was added, and then incubated at room temperature for 15 min. One-hundred and fifty ml of the sample extracts and 150 ml of Assay 2 3 Buffer which contains the substrate o-nitrophenyl-b-galactoside were mixed and incubated at 37°C for 30 min. The absorbance at 420 nm was read with a spectrophotometer to detect o-nitrophenol, which is yellow. The absorbance values of the reference untreated samples were used as control each time. The resulting values of b-galactosidase activity were normalized to milli unit (mu) of b-galactosidase per mg of protein. One unit of b-galactosidase hydrolyzes 1 micromole of o-nitrophenyl-b-D-galactopyranoside (ONPG) to o-nitrophenol and galactose per minute at pH 7.5 and 37°C. A quantity of sample protein was assayed with Bio-Rad Protein Assay Kit (Bio-Rad). In brief, 50 ml of the sample extracts and 2.5 ml of dye solution were mixed and incubated at room temperature for 5 min. The absorbance at 595 nm was read with a spectrophotometer. Transgene expression in the progeny by Northern blot analysis. For northern blot analysis, the total RNAs were extracted from the whole bodies of the progeny by using ISOGEN (Nippongene). Each RNA sample (20 mg) was separated on a 1% agarose denatured gel, transferred to a NitroPlus nitrocellulose membrane (Micron Separations Inc.), and the blot was hybridized with the 32P-labelled lacZ fragment. The blot was autoradiographed at 270°C for 7 days. Electron microscopic analysis. Negative stained electron micrographs of cationic liposomes were studied on a JEM-1010 transmission electron microscope (JEOL Ltd., Tokyo, Japan). To prepare the liposomes for visualization, a drop of liposome solution was added to 400 mesh copper grids coated with a formvar/carbon support film.
The excess solution was absorbed with Watman filter paper (standard 50). A drop of phosphotungstic acid solution (2% w/v) was then placed on the grid, and immediately absorbed with Watman filter paper. The grid was then dried for 5 min under ambient conditions before microscopic examination. Analysis of organ toxicity. Serum from animals before and after the administration of cationic liposome was obtained and subjected to biochemical analyses by assaying the levels of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). Organ specimens, including liver from cationic liposomeadministered mice and those without the administration of liposome were obtained from maternal or neonatal animals, fixed in formaline, embedded in paraffin, and stained in hematoxylin and eosin. Representative sections were examined for cytotoxicity.
RESULTS Cationic Lipids with Enhanced Gene Transfer into Animals The pCMV z SPORT-bgal expression plasmid was used to assess the gene transfer efficiency of a number of different cationic liposomes in vivo. DNA/liposome complex was administered into pregnant animals at day 11.5 p.c.. To confirm the presence of plasmid DNA sequences, animals were sacrificed two days after the administration and then the genomic DNAs were extracted from embryos and maternal liver and subjected to Southern blot analysis. Among 8 commercialized cationic liposomes that were tested, DMRIE-C reagent allowed maximum transduction of the genes into maternal liver (Fig. 1a) as well as fetus (Fig. 1b). We tested fetal gene transfer using different batches of DMRIE-C reagent (No. FK902, HCM903 and JHYY02). Among tested pregnant ICR mice at day 11.5 p.c., Southern analysis showed that 20 out of 26 aminals were positive for fetal gene transfer. There was no significant difference between the three batches (data not shown). The amounts of transferred gene were different by individuals and organs: 100-1650 pg/10 mg of total DNA obtained from maternal organs; 0.1-10.0 pg/10 mg of total DNA from fetuses. In treated mice, we detected supercoiled and linearized forms of intact pCMV z SPORT-bgal plasmid DNA in maternal liver. In contrast, only a linearized form of plasmid DNA was detected in fetuses. Despite a small amount of the DNA, cationic liposomes with Gene Transfer and ExGen 500 allow gene transfer into maternal liver, whereas no gene transfer was detected by any other liposomes (Fig. 1a). By Southern analysis, the plasmid DNA was detected only in DNA samples from fetuses administered with DMRIE-C reagent (Fig. 1b). Although we do not show the results, other liposomes such as FuGene 6 (Boehringer Mannheim), Effectene Transfection Reagent (Qiagen Inc.) and LipofectAMINE plus (Life Technologies Inc.) formed large aggregates soon after making the mixture with plasmid DNA, and caused not be used. Another 15 non-commercialized liposomes including those with different molar ratios of DOTMA/
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FIG. 2. Optimization of the concentration of DMRIE-C and plasmid DNA. Different DMRIE-C amounts at 10 mg, 100 mg, 500 mg, and 1000 mg were mixed with the plasmid DNA which was held constant at 100 mg. Efficiency was monitored by evaluating b– galactosidase gene expression in the liver 48 hr after the administration of the complexes into adult female mice (number of animals tested in each group 5 4).
DOPE, TMAG/DLPC/DOPE showed no positive results for fetal gene transfer. These results suggest that DMRIE-C reagent is one of the most suitable cationic liposomes for in vivo gene transfer among the tested 26 liposomes. Optimization of DMRIE-C and Plasmid DNA for in Vivo Study To examine the effect of the concentration of DMRIE-C and plasmid DNA complex on gene transfer in vivo, different DMRIE-C concentrations were tested, 10 mg, 100 mg, 500 mg and 1000 mg, whereas the plasmid DNA was held constant at 100 mg. Efficiency was monitored by evaluating b– galactosidase gene expression in the liver 48 hr after the administration of the complexes into adult mice with 4 animals being used for each group. As shown in Fig. 2, use of 500 mg of DMRIE-C reagent resulted in maximal efficiency. Therefore, for the following studies we decided that the concentration of DMRIE-C suitable for in vivo administration into mice was 500 mg when 100 mg plasmid DNA was used. Distribution of the Transferred Gene in Vivo Next we analyzed the organ distribution and expression of the transferred gene with DMRIE-C complexes in adult animals. Forty eight hours after the administration of DMRIE-C/DNA complexes into normal ICR male and female mice, animals were sacrificed and the genomic DNA and protein were extracted from several organs. Southern analysis showed that the transferred gene was detected in a broad spectrum of organs including lung, heart, liver, kidney, spleen, pancreas, stomach, large intestine and small intestine. The amounts of DMRIE-C/DNA complexes were high in the liver. This observation is in agreement with the other results of a tissue distribution of plasmid DNA/DMRIE
complex following the intravenous administration into mice (21, 22). We could not detect the transferred gene in other organs such as brain, testis and ovary. In the treated mice, we detected supercoiled and linearized forms of intact pCMV z SPORT-bgal plasmid DNA in maternal lung and liver. In contrast, only a linearized form of plasmid DNA was detected in other organs. To evaluate the form of the transferred plasmid DNA in fetuses, we used ATP-dependent deoxyribonuclease (ADD) which specifically recognizes and digests linear molecule. The result showed that the transferred DNA was digested completely with ADD, indicating that the form of plasmid DNA in fetuses is a linear molecule. These data are summarized in Table 1. Southern analysis showed that the transferred gene was present for 4 to 6 days after the administration and degraded thereafter (data not shown). Whether another DMRIE reagent conjugated with DOPE (22, 23) or without cholesterol (21) is valid for fetal gene transfer will have to await further analysis. Expression Profiles of the Transferred Gene in Adult Mice Mice were studied for b– galactosidase gene expression following administration of 100 mg pCMV z SPORT-bgal plasmid DNA complexed with DMRIE-C reagent. In the treated mice, reporter gene activity was detected in several organs in which plasmid DNA was recognized (Fig. 3). The b– galactosidase activity per milligram of tissue protein of lung and liver showed the highest levels among organs of adult mice. No reporter gene activity was detected in any other organs which
TABLE 1
Schematic Summary of Distribution of the Transferred Gene in Vivo Plasmid form Organ
Number of copies/cell
Open circle
Linear
Supercoil
Brain Heart Lung Liver Kidney Spleen Pancreas Stomach Colon Small intestine Testis Ovary
0 2–5 20–50 10–40 2–10 2–5 2–10 1–2 0–2 0–2 0 0
2 2 1 1 2 2 2 2 2 2 2 2
2 1 1 1 1 1 1 1 1 1 2 2
2 2 1 1 2 2 2 2 2 2 2 2
Note. Seven-week-old ICR male and female mice (n 5 12) were injected i.v. with 100 mg pCMV z SPORTbgal plasmid DNA conjugated with 500 mg DMRIE-C reagent. Two days later, several organs were obtained, and the genomic DNAs from them were subjected to Southern blot analysis for the presence of transferred gene.
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istration (Fig. 4b). Although we did not show the individual results, gene expression of the transferred gene occurred equally in every fetus recovered from a dam. These results suggest that our method can successfully transfer the administered gene into a fetus and allows the gene expression transiently. The Structural Features of the DMRIE-C Reagent
FIG. 3. Transgene expression in adult mice. Mice were studied for b– galactosidase gene expression following i.v. administration of 100 mg pCMV z SPORT-bgal palsmid DNA conjugated with DMRIE-C reagent. The b– galactosidase activity per milligram of tissue protein of several organs was demonstrated (number of animals tested in each organ 5 4).
The structural characteristics of the DMRIE-C cationic liposome were studied. Electron microscopy (EM) studies have revealed that DMRIE-C (batch HCM903) forms a particle 250-500 nm in diameter (Fig. 5a) and shows multilamellar vesicles (Fig. 5c). Notable conformational changes were not detected when the DMRIE-C reagent was conjugated with plasmid DNA (Fig. 5b). These results suggest that the DMRIE-C reagent maintains the original size and feature after being complexed with DNA. It should be emphasized that the DMRIE-C/DNA complexes form no large aggregates even in the presence of serum. In contrast,
have no transferred gene. No b– galactosidase activity was detected in any of the untreated controls. Although data are not shown, expression of the reporter gene in organs of adult mice lasted up to 12 days in lung and liver, 6 days in kidney, spleen and heart, and 4 days in stomach, colon and intestine after gene transfer using DMRIE-C reagent in vivo. To evaluate the positivity of the gene transfer in vivo, tissue sections were prepared from adult liver administered with DMRIE-C/DNA complexes and then analyzed for the gene expression by b-galactosidase histochemical staining. Despite the very low number of cells, positive signals were detected in the liver (approximately 0.1 to 2.0%), whereas no positive staining was detected in the untreated liver tissues. Characteristic features of many of these cells in the liver include polygonal cellular morphology, centrally placed uniform nuclei, and localization in the hepatic plate, elucidating them as typical hepatocytes. Although the percentage of positive cells varied, positive signals were also detected in heart, lung, spleen, kidney, pancreas, stomach, colon and intestine. Gene Expression of the Progeny We next assessed the b-galactosidase gene expression in the fetuses when DMRIE-C/DNA complexes were injected at day 11.5 p.c. The proteins and total RNAs from the fetus were extracted periodically, and subjected to b-galactosidase assay and northern blot analysis, respectively. The b-galactosidase activity was detected up to 4 days after administration (Fig. 4a). The b-gal activity of the fetus at 2 days after administration was the highest of all the samples. The b-galactosidase gene transcripts were clearly detected in RNA samples from the progeny 2 days after admin-
FIG. 4. Transgene expression in the progeny. (a) After the administration of Liposome/DNA complexes into pregnant mice at day 11.5 p.c. (n 5 4), the protein from the fetus was extracted periodically at 1, 2, 3, 4 and 6 days, and was then subjected to b– galactosidase assay. (b) Two days after the administration, total RNAs were extracted from the progeny, and were then subjected to northern blot analysis by using a 32P-labeled b– galactosidase DNA fragment as a probe. Lanes 1 and 2, fetuses that received DMRIE-C and plasmid DNA; lanes 3 and 4, untreated fetuses. As a reference, transcripts of b–actin gene were analyzed.
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FIG. 5. The structural study of the liposome/DNA complex. Negative stained electron micrographs of cationic liposomes were studied on a JEM-1010 transmission electron microscope. (a) DMRIE-C alone; (b) DMRIE-C/DNA complex at optimized ratio; (c) large magnitude of DMRIE-C particle; (d) DOGS alone; (e) DOGS/DNA complex at a ratio of 1 mg DNA to 6 nmol DOGS.
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DOGS (batch 6414) forms particle 150-300 nm in diameter (Fig. 5d) and showed a conformational changes with many aggregates when mixed with plasmid DNA (Fig. 5e). In Vivo Toxicity of Cationic Liposome-DNA Conjugates The effects of intravenous cationic liposome on maternal liver were assessed by measurement of serum GOT and GPT levels. The serum GOT and GPT levels in adult animals 2 days after a single intravenous injection of 0.5 ml PBS(2) containing 500 mg of DMRIE-C reagent and 100 mg of pCMV z SPORT-bgal plasmid DNA into day 11.5 p.c. pregnant mice were 88 6 12 IU/liter and 40 6 7 IU/liter, respectively (n 5 4). Analysis of serum enzymes before the treatment revealed that the serum GOT and GPT levels were 86 6 14 IU/liter and 34 6 7 IU/liter, respectively (n 5 4), indicating there were no significant changes before and 2 days after the administration. Histopathology analysis of organs (brain, liver, spleen, lung, kidney) from these animals showed no notable abnormalities in the early phase of postadministration (data not shown). These results of the pathological and biochemical examinations demonstrated that the administration of cationic liposome/DNA complex used in this study did not show significant toxicity to animals. DISCUSSION In this study, we evaluated cationic liposome formulations suitable for systemic in vivo gene transfer into animals. We found that a new cationic lipid, DMRIE-C reagent, resulted in successful gene delivery and expression in a variety of murine organs. Despite low efficiencies when compared to dams, we also observed the DMRIE-C/DNA complexes allowed gene transfer and expression in fetuses after i.v. injection into pregnant mice at day 11.5 p.c.. When we injected the DMRIE-C/DNA complexes at day 15.5 p.c. into pregnant animals, gene transfer was observed in the neonates (Takahama et al., in preparation). No significant biochemical abnormalities or organ toxicities were observed following intravenous injection of DMRIE-C/ DNA complexes. Although we do not know the precise mechanisms of the transplacental spread of the gene, the placenta may have some important role in the delivery of biologically active factors to the developing embryos (24). This new cationic liposome formulation should provide an attractive model system to deliver biologically significant genes into animals systemically in order to look at the effect of these genes in vivo. Based on electron microscopic analysis, the DMRIE-C reagent showed multilamellar vesicles with a relatively large particle size of 250-500 nm in diameter. Although the sort of vesicles most suitable for in vivo
gene transfer is not well defined, our study showed that efficient systemic gene delivery was attained when cationic liposomes formed multilamellar vesicles. It is necessary to identify more effective cationic liposomes by synthesizing a large number of structurally related molecules and to monitor them in an appropriate in vivo assay system. Besides liposome formulation, another key factor for cationic lipid-based gene transfer in vivo is the conformational changes of liposome after complexed to DNA (1). Electron microscopic data show that addition of DMRIE-C to the plasmid DNA caused no greater changes of liposome in the mean particle diameter. Furthermore, it is proposed that ineffective gene transfer of cationic liposomes in vitro is brought about by the ready formation of a large aggregate among liposome/DNA complexes (25). In contrast, our results indicated that the complex of plasmid DNA and DMRIE-C formed less aggregation even in the blood when it was compared to other lipids such as DOGS. Considering that the large amounts of visible aggregates of DOGS/DNA complexes were mainly trapped in the lung of dams, which may reduce the gene transfer efficiency into fetuses, our success in systemic gene transfer with the DMRIE-C reagent may be partly due to a less aggregative nature of cationic lipid/DNA complexes. It is also possible that the liposome/DNA complex could exert a toxic effect in vivo. However, we did not observe significant toxicity when examining murine GOT and GPT levels after the administration of liposome/DNA complexes. In addition, no abnormalities in histopathology of major organs were present in the treated mice after 2 weeks. Thus, the DMRIE-C reagent did not show significant toxicity at the indicated conditions. In view of the safety concerns in fetal gene therapy (26), it is necessary to achieve an efficient gene delivery into fetuses without permanent integration of the gene into host chromosomes, which may in turn be passed on to the following generations. Our first report on the use of DOGS showed the transient expression of the introduced genes into fetuses without any integration of the genes into germ cells. Although we did not examine the germ line transmission of the animals after the administration of the DMRIE-C/DNA complexes, it is unlikely that they facilitated integration of the gene in vivo. Specific organ targeting in vivo might become a basis for an ideal gene therapy (27-30). Targeting to the appropriate organs will be achieved using surgical procedures, receptor-targeted uptake or a gene regulatory element such as a tissue-specific promoter/enhancer system. Interestingly, DMRIE-C/DNA complexes tend to accumulate especially in the liver and lung of animals. Although the reason for high accumulation of the liposome-DNA complex in specific organs is not fully understood, the lipid-to-plasmid charge ratio may cause organ trapping of the complex (11). Further studies are required to determine whether our method for
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fetal gene transfer is applicable for fetal organ-specific gene delivery and expression. Fetal gene therapy appears to be an ideal method to rescue embryonal dysfunction and early organ damage as the result of inherited genetic diseases (31). Recently, after our transplacental delivery with cationic liposome/DNA injection into the tail vein of pregnant mice (13), several in vivo attempts were made at gene transfer into fetuses. Pitt et al. have transduced recombinant retroviruses into the airways of fetal sheep in utero (32). A similar approach was used by McCry et al. to transfer adenovirus vectors carrying the human CFTR gene to the fetal ovine airways (33). In addition, retroviral infected fetal rat intestine was grafted subcutaneously into immune-deficient mice (34). As another interesting approach, intra-amniotic gene administration was also tested in an in vitro (35) and in vivo (36, 37) system by using cationic lipid mediated transfection, hemagglutinin virus of Japan (HVJ)liposome method, retroviral and adenoviral infection. Our nonviral cationic liposome transfection has certain advantages over these methods: (1) a simple and easy method for administration which does not require surgical intervention; (2) non-integrating vector system; (3) non-toxic and possibly non-immunogenic reagent. A major problem of cationic liposome is that liposome change its character from batch to batch and the reproducibility of the method became sometimes problematic. In this regards, gene transfer by the use of DMRIE-C as described here was reproducible when different batches were used and different scientists used the procedure. The method will be useful in analyzing the function of several genes which are important in embryonal development by introducing these genes at desired stages and into organs of embryogenesis. ACKNOWLEDGMENTS We thank Mr. T. Komatsu for animal care and Ms. M. Abe for technical assistance. This work was supported in part by a Grantin-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, by a Grantin-Aid for Cancer Research from the Ministry of Health and Welfare in Japan, and by the Bristol-Myers Squibb Foundation. Y. Takahama was awarded a Research Resident Fellowship from the Foundation for Promotion of Cancer Research.
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6. Lee, E. R., Marshall, J., Siegel, C. S., et al. (1996) Hum. Gene Ther. 7, 1701–1717. 7. Felgner, P. L. (1996) Hum. Gene Ther. 7, 1791–1793. 8. Balasubramanian, R. P., Bennett, M. J., Aberle, A. M., et al. (1996) Gene Ther. 3, 163–172. 9. Thierry, A. R., Lunardi-Iskander, T., Bryant, J. L., et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9742–9746. 10. Thierry, A. R., Rabinovich, P., Peng, B., et al. (1997) Gene Therapy 4, 226 –37. 11. Mahato, R. I., Anwer, K., Tagliaferri, F., et al. (1998) Hum. Gene Ther. 9, 2083–2099. 12. Behr, J-P., Demeneix, B., Loeffler, J-P., et al. (1989) Proc. Natl. Acad. Sci. USA 86, 6982– 6986. 13. Tsukamoto, M., Ochiya, T., Yoshida, S., Sugimura, T., and Terada, M. (1995) Nature Genetics 9, 243–248. 14. Felgner, J. H., Kumar, R., Sridhar, C. N., et al. (1994) J. Biol. Chem. 269, 2550 –2561. 15. Felgner, P. L., et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7413–7417. 16. Hawley-Nelson, P., Ciccarone, V., Gebeyehu, G., et al. (1993) Focus 15, 73–79. 17. Rose, J. K., Buonocore, L., and Whitt, M. A., (1991) Biotechniques 10, 520 –525. 18. Logan, J. J., Bebock, Z., Walker, L. C., et al. (1995) Gene Ther. 2, 38 – 49. 19. Yagi, K., Noda, H., Kurono, M., and Ohishi N. (1993) Biochem. Biophis. Res. Commun. 196, 1042–1048. 20. Boussif, O., Lezoualc’h, F., Zanta, M-A., et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7297–7301. 21. Oku, N., Tokudome, Y., Namba, Y., et al. (1996) Biochem. Biophys. Acta 1280, 149 –154. 22. Parker, S. E., Ducharme, S., Norman, J., et al. (1997) Hum. Gene Ther. 8, 393– 401. 23. Hsiao, M., Tse, V., Carmel, J., et al. (1997) Biochem. Biophys. Res. Commun. 233, 359 –364. 24. Cross, J.C., Werb, Z., and Fisher, S.J. (1994) Science 266, 1508 – 1518. 25. Jaaskelainen, I., Monkkonen, J., and Urtti, A. (1994) Biochim. Biophys. Acta 1195, 115–123. 26. Smith, K. T., Shepherd, A. J., Boyd, J. E., and Lees, G. M. (1996) Gene Ther. 3, 190 –200. 27. Perales, J. C., Ferkol, T., Beegen, H., and Rantoff, O. D. (1994) Proc. Natl. Acad. Sci. USA 91, 4086 – 4090. 28. Trubeskoy, V. S., Torchilin, V. P., Kennel, S., and Huang, L. (1992) Biochim. Biophys. Acta 1131, 311–313. 29. Marck, K. D., Rosemary, W., and Zeldis, J. B. (1994) Am. J. Med. Sci. 307, 138 –143. 30. McLachlan, G., Davidson, D. J., Stevenson, B. J., et al. (1995) Gene Ther. 2, 614 – 622. 31. Coutelle, C., Douar, A-M., Colledge, W. H., and Froster, U. (1995) Nature Med. 1, 864 – 866. 32. Pitt, B. R., et al. (1995) Gene Ther. 2, 344 –350. 33. McCray, P. B., et al. (1995) J. Clin. Invest 95, 2620 –2632. 34. Jacomino, M., Lau, C., James, S. Z. et al. (1996) Hum. Gene Ther. 7, 1757–1762. 35. Douar, A-M., Themis, M., Sandig, V., et al. (1996) Gene Ther. 3, 789 –796. 36. Harmanjatinder, S. Sekhon, S., and Larson, J. E. (1996) Nature Med. 1, 1201–1203. 37. Hayashi, S-I., Morishita, R., Aoki, M., et al. (1996) Gene Ther. 3, 878 – 885.
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Evaluation of Cationic Liposome Suitable for Gene Transfer into Pregnant Animals Takahiro Ochiya,* Yasushi Takahama,* Hiroyasu Baba-Toriyama,† Makoto Tsukamoto,‡ Yuko Yasuda,§ Hiroshi Kikuchi, ¶ and Masaaki Terada* ,1 *Genetics Division and †Chemotherapy Division, National Cancer Center Research Institute, Tokyo, Japan; ‡Internal Medicine, Chiba University School of Medicine, Chiba, Japan; §Louis Pasteur Center for Medical Research, Kyoto, Japan; and ¶Daiichi Seiyaku Co., Ltd. Research Institute, Tokyo, Japan
Received March 25, 1999
Cationic liposome-mediated in vivo gene transfer represents a promising approach for somatic gene therapy. To assess the most suitable liposome for gene delivery into a wide range of organs and fetuses in mice, we have explored several types of cationic liposomes conjugated with plasmid DNA carrying the b-galactosidase gene through intravenous injection into pregnant animals. Transduction efficiency was assessed by Southern blot analysis and expression of the transferred gene was evaluated by enzymatic demonstration of b-galactosidase activity. Through the analysis of several types of recently synthesized cationic liposome/lipid formulations, DMRIE-C reagent, a liposome formulation of the cationic lipid DMRIE (1,2dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) and cholesterol in membrane-filtered water met our requirements. When the plasmid DNA/ DMRIE-C complexes were administered intravenously into pregnant mice at day 11.5 post coitus (p.c.), transferred genes were observed in several organs in dams and were expressed. Furthermore, although the copy numbers transferred into embryos were low, we observed reporter gene expression in the progeny. © 1999 Academic Press
Key Words: cationic liposome; gene therapy; fetus.
Recently, nucleic acids are delivered to cells by transduction with viral particles or by transfection which includes chemical or physical methods. Cationic liposomes have been used mostly in in vitro gene transfer experiments because they offer several advantages over other chemical means, such as calcium phosphate coprecipitation or DEAE-Dextran (1, 2, 3). These advantages include: (1) non-viral delivery system mini1 To whom correspondence should be addressed. National Cancer Center Research Institute 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo, 104 Japan. Fax: 81-3-3541-2685. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
mizing concerns related to public health and vector safety; (2) availability to produce transient and stable transfection in vitro; (3) ability to transfer a variety of macromolecules, including several types of DNA with plasmid, oligonucleotide and yeast artificial chromosome, RNA and protein; (4) ease of large quantity liposome production; (5) repeatable administration in vivo; (6) protection of plasmid DNA molecules from degradation by nucleases and shearing by aerosolisation; (7) relatively low cytotoxicity for mammalian cells. Several features of these nonviral vectors are attractive for gene transfer studies in vivo, including their relative safety and lack of toxicity (4-11). However, a limitation of these cationic liposomes has been a relative inefficiency of gene transfer in vivo. We previously reported that a single intravenous (i.v.) injection of foreign gene-cationic lipopolyamine containing dioctadecylamido-glycylspermine (DOGS) (12) complexes into pregnant mice enables transgenes to be expressed in the fetuses and postpartum progeny (13). However, we and others later noticed that the efficiency of the gene transfer into fetuses is varied and the batches of DOGS currently available allowed no positive results. We do not know the reason why we have had difficulty in reproducing with consistent efficiency a gene transfer by our previous method by using different batches of DOGS. But, we have observed that the DNA/DOGS complex often made a large number of aggregates when current batches of DOGS were used. The aggregates may be trapped in the lung, which in turn produced an unsuccessful gene transfer into the fetus. The extent of the aggregates may depend on the purity of plasmid DNA, incubation time, temperature, and the liposome batch and differ from experiment to experiment. Studies were performed to determine optimal cationic liposome formulation for successful gene transfer into adult animals or progeny of the pregnant animals. The safety and toxicity of those lipid
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FIG. 1. Systemic gene delivery of plasmid DNA to pregnant animals. The pCMV z SPORT-bgal expression plasmid was used to assess the gene transfer efficiency of a number of different cationic liposomes in vivo. Liposome/DNA complex was administered into pregnant animals at day 11.5 p.c. Animals were sacrificed two days after the administration and then the genomic DNAs were extracted from embryos and maternal liver and subjected to Southern blot analysis. Among 8 cationic liposomes that were tested, DMRIE-C reagent allowed maximum transduction of the genes into maternal liver (Fig. 1a) as well as fetus (Fig. 1b). Each lane contained 10 mg of undigested genomic DNA. As a positive control, 100 pg of uncut (supercoiled) and linealized plasmid DNA were loaded.
formulations were also assessed in vivo after systemic i.v. injection into mice. Here, we show that the DMRIE-C reagent allows a reproducible and an efficient systemic delivery of genes into several organs of dams and allows expression of the transferred genes. In their progeny, although in a small amount, we observed gene transduction and expression. MATERIALS AND METHODS Plasmid expression vector and cationic lipids. An eukaryotic expression vector plasmid containing b-galactosidase gene, pCMV z SPORT-bgal (Life Technologies Inc.), was used for all the experiments. The plasmid DNA was purified through Wizard Plasmid Purification System (Promega), ethanol-precipitated twice, and finally dissolved in high quality, nuclease-free water. Cationic lipid was purchased: Transfectam (12) (DOGS: dioctadecyldimethylammonium chloride) and Tfx-50 (14) (cationic lipid [N,N,N9,N9-tetramethyl-N,N9-bis(2-hydroxy-ethyl)-2,3,-dioleoyloxy1,4-butanediammonium iodide]/DOPE: dioleoylphosphatidylethanolamine) from Promega, USA; Lipofectin (15) (DOTMA: N-(2,3(dioleoyloxy)propyl)-N,N,N-trimethyl ammonium chloride/DOPE: dioleoylphosphatidylethanolamine), LipofectAMINE (16) (DOSPA: 2,3-dioeoyloxy-N-(2(spermidinecarboxamodo)-ethyl)-N,N-dimethyl-
1-propanaminium trifluoroacetate/DOPE), LipofectAce (17) (DDAB: dimethyldioctadecylammonium bromide/DOPE) and DMRIE-C (18) (DMRIE: 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide/cholesterol) from Life Technologies Inc.; Gene Transfer (19) (TMAG: a-(trimethylammonioacetyl)-didodecyl D-glutamete chloride/DLPC: dilauroyl phosphatidylcholine/DOPE) from Wako, Japan; ExGen 500 (20) (cationic polymer polyethylenimine) from Euromedex. To determine the optimal ratio of cationic liposome to plasmid DNA, pCMV z SPORT-bgal was complexed with indicated liposomes at increasing amounts of 10 mg to 1000 mg whereas the DNA was held constant at 100 mg. Other non-commercialized liposomes including different molar ratios of DOTMA/DOPE, TMAG/ DLPC/DOPE were generously gifted from Daiichi Seiyaku Co. Ltd. Research Institute. In total, 26 cationic liposomes were tested. In vivo gene transfer. Male and female adult mice (7-week old) and pregnant ICR mice at day 11.5 p.c. were used for in vivo experiments. The DNA/liposome complex for in vivo administration was prepared as follows. A 250 ml solution of cationic liposome containing 10-1000 mg of DMRIE-C reagent was added to 250 ml of PBS(2) containing 100 mg of plasmid DNA drop by drop (not the reverse order), then mixed gently by drawing and withdrawing the solution into and out of the syringe three to five times. A cloudy solution may appear. The mixed solution was incubated at room temperature for 6 to 7 min. Incubation for more than 8 min leads to excess aggregates which may result in a less efficient transfer
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of the gene. Just before administration into animals, the solution was mixed by inverting the tube 2-3 times. The mixed solution should not have visible aggregates. The entire solution was then drawn into a disposable 1 ml plastic syringe fitted with a 27G 33/4 needle, and injected into the tail vein of adult male and female animals and pregnant ICR mice at day 11.5. The animals were lightly anesthetized when given intravenous injections, and the injection speed was about 0.5 ml/4-5 seconds. Concerning other liposomes tested in this experiment, the DNA and lipid were each diluted to 0.25 ml with PBS(2) and then mixed by adding the liposome solution to the DNA drop by drop. The optimal ratios of DNA to liposomes (wt/wt) are: DOGS, 1:5; Tfx-50, 1:5; Lipofectin, 1:10; LipofectAMINE, 1:5; LipofectAce, 1:10; Gene Transfer, 1:5; ExGen 500, 1:10; DOTMA/DOPE, 1:5; TMAG/DLPC/DOPE, 1:5, which were determined by in vitro transfection analysis by the use of primary cultures from fetal mice fibroblasts (unpublished observations). The DNA/liposome complexes from Lipofectin, LipofectAMINE and LipofectAce were administered immediately into mice. The other complexes from DOGS, Tfx-50, Gene Transfer, DOTMA/DOPE, TMAG/DLPC/ DOPE and ExGen 500 were incubated 5 to 10 min at room temperature and then used. Southern analysis for the detection of the transgene. The genomic DNA from whole embryo bodies and their maternal organs was subjected to Southern blot analysis for the presence of the b-galactosidase gene. Ten mg of genomic DNA was separated by electrophoresis on a 0.8% agarose gel, transferred onto a nylon membrane (Hybond N plus, Amersham), and hybridized with a a 32 P-labeled b-galactosidase fragment DNA as a probe in 5 3 Denhardt’s solution (1 3 Denhardt’s 5 0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 0.5% SDS, 5 3 sodium saline phosphate EDTA (1 3 sodium saline phosphate EDTA 5 150 mM NaCl, 10 mM sodium phosphate, 1.3 mM EDTA), and 200 mg/ml of salmon testis DNA at 65°C for 16 hr. The filters were then washed in 0.1% SDS and 0.1 3 sodium saline phosphate EDTA at 65°C. Assay of b-galactosidase activity in vitro. b-galactosidase activity was measured with b-Galactosidase Enzyme Assay System (Promega). In brief, homogenized tissues were washed with PBS(2) several times, and 1 ml of 1x Reporter Lysis Buffer was added, and then incubated at room temperature for 15 min. One-hundred and fifty ml of the sample extracts and 150 ml of Assay 2 3 Buffer which contains the substrate o-nitrophenyl-b-galactoside were mixed and incubated at 37°C for 30 min. The absorbance at 420 nm was read with a spectrophotometer to detect o-nitrophenol, which is yellow. The absorbance values of the reference untreated samples were used as control each time. The resulting values of b-galactosidase activity were normalized to milli unit (mu) of b-galactosidase per mg of protein. One unit of b-galactosidase hydrolyzes 1 micromole of o-nitrophenyl-b-D-galactopyranoside (ONPG) to o-nitrophenol and galactose per minute at pH 7.5 and 37°C. A quantity of sample protein was assayed with Bio-Rad Protein Assay Kit (Bio-Rad). In brief, 50 ml of the sample extracts and 2.5 ml of dye solution were mixed and incubated at room temperature for 5 min. The absorbance at 595 nm was read with a spectrophotometer. Transgene expression in the progeny by Northern blot analysis. For northern blot analysis, the total RNAs were extracted from the whole bodies of the progeny by using ISOGEN (Nippongene). Each RNA sample (20 mg) was separated on a 1% agarose denatured gel, transferred to a NitroPlus nitrocellulose membrane (Micron Separations Inc.), and the blot was hybridized with the 32P-labelled lacZ fragment. The blot was autoradiographed at 270°C for 7 days. Electron microscopic analysis. Negative stained electron micrographs of cationic liposomes were studied on a JEM-1010 transmission electron microscope (JEOL Ltd., Tokyo, Japan). To prepare the liposomes for visualization, a drop of liposome solution was added to 400 mesh copper grids coated with a formvar/carbon support film.
The excess solution was absorbed with Watman filter paper (standard 50). A drop of phosphotungstic acid solution (2% w/v) was then placed on the grid, and immediately absorbed with Watman filter paper. The grid was then dried for 5 min under ambient conditions before microscopic examination. Analysis of organ toxicity. Serum from animals before and after the administration of cationic liposome was obtained and subjected to biochemical analyses by assaying the levels of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). Organ specimens, including liver from cationic liposomeadministered mice and those without the administration of liposome were obtained from maternal or neonatal animals, fixed in formaline, embedded in paraffin, and stained in hematoxylin and eosin. Representative sections were examined for cytotoxicity.
RESULTS Cationic Lipids with Enhanced Gene Transfer into Animals The pCMV z SPORT-bgal expression plasmid was used to assess the gene transfer efficiency of a number of different cationic liposomes in vivo. DNA/liposome complex was administered into pregnant animals at day 11.5 p.c.. To confirm the presence of plasmid DNA sequences, animals were sacrificed two days after the administration and then the genomic DNAs were extracted from embryos and maternal liver and subjected to Southern blot analysis. Among 8 commercialized cationic liposomes that were tested, DMRIE-C reagent allowed maximum transduction of the genes into maternal liver (Fig. 1a) as well as fetus (Fig. 1b). We tested fetal gene transfer using different batches of DMRIE-C reagent (No. FK902, HCM903 and JHYY02). Among tested pregnant ICR mice at day 11.5 p.c., Southern analysis showed that 20 out of 26 aminals were positive for fetal gene transfer. There was no significant difference between the three batches (data not shown). The amounts of transferred gene were different by individuals and organs: 100-1650 pg/10 mg of total DNA obtained from maternal organs; 0.1-10.0 pg/10 mg of total DNA from fetuses. In treated mice, we detected supercoiled and linearized forms of intact pCMV z SPORT-bgal plasmid DNA in maternal liver. In contrast, only a linearized form of plasmid DNA was detected in fetuses. Despite a small amount of the DNA, cationic liposomes with Gene Transfer and ExGen 500 allow gene transfer into maternal liver, whereas no gene transfer was detected by any other liposomes (Fig. 1a). By Southern analysis, the plasmid DNA was detected only in DNA samples from fetuses administered with DMRIE-C reagent (Fig. 1b). Although we do not show the results, other liposomes such as FuGene 6 (Boehringer Mannheim), Effectene Transfection Reagent (Qiagen Inc.) and LipofectAMINE plus (Life Technologies Inc.) formed large aggregates soon after making the mixture with plasmid DNA, and caused not be used. Another 15 non-commercialized liposomes including those with different molar ratios of DOTMA/
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FIG. 2. Optimization of the concentration of DMRIE-C and plasmid DNA. Different DMRIE-C amounts at 10 mg, 100 mg, 500 mg, and 1000 mg were mixed with the plasmid DNA which was held constant at 100 mg. Efficiency was monitored by evaluating b– galactosidase gene expression in the liver 48 hr after the administration of the complexes into adult female mice (number of animals tested in each group 5 4).
DOPE, TMAG/DLPC/DOPE showed no positive results for fetal gene transfer. These results suggest that DMRIE-C reagent is one of the most suitable cationic liposomes for in vivo gene transfer among the tested 26 liposomes. Optimization of DMRIE-C and Plasmid DNA for in Vivo Study To examine the effect of the concentration of DMRIE-C and plasmid DNA complex on gene transfer in vivo, different DMRIE-C concentrations were tested, 10 mg, 100 mg, 500 mg and 1000 mg, whereas the plasmid DNA was held constant at 100 mg. Efficiency was monitored by evaluating b– galactosidase gene expression in the liver 48 hr after the administration of the complexes into adult mice with 4 animals being used for each group. As shown in Fig. 2, use of 500 mg of DMRIE-C reagent resulted in maximal efficiency. Therefore, for the following studies we decided that the concentration of DMRIE-C suitable for in vivo administration into mice was 500 mg when 100 mg plasmid DNA was used. Distribution of the Transferred Gene in Vivo Next we analyzed the organ distribution and expression of the transferred gene with DMRIE-C complexes in adult animals. Forty eight hours after the administration of DMRIE-C/DNA complexes into normal ICR male and female mice, animals were sacrificed and the genomic DNA and protein were extracted from several organs. Southern analysis showed that the transferred gene was detected in a broad spectrum of organs including lung, heart, liver, kidney, spleen, pancreas, stomach, large intestine and small intestine. The amounts of DMRIE-C/DNA complexes were high in the liver. This observation is in agreement with the other results of a tissue distribution of plasmid DNA/DMRIE
complex following the intravenous administration into mice (21, 22). We could not detect the transferred gene in other organs such as brain, testis and ovary. In the treated mice, we detected supercoiled and linearized forms of intact pCMV z SPORT-bgal plasmid DNA in maternal lung and liver. In contrast, only a linearized form of plasmid DNA was detected in other organs. To evaluate the form of the transferred plasmid DNA in fetuses, we used ATP-dependent deoxyribonuclease (ADD) which specifically recognizes and digests linear molecule. The result showed that the transferred DNA was digested completely with ADD, indicating that the form of plasmid DNA in fetuses is a linear molecule. These data are summarized in Table 1. Southern analysis showed that the transferred gene was present for 4 to 6 days after the administration and degraded thereafter (data not shown). Whether another DMRIE reagent conjugated with DOPE (22, 23) or without cholesterol (21) is valid for fetal gene transfer will have to await further analysis. Expression Profiles of the Transferred Gene in Adult Mice Mice were studied for b– galactosidase gene expression following administration of 100 mg pCMV z SPORT-bgal plasmid DNA complexed with DMRIE-C reagent. In the treated mice, reporter gene activity was detected in several organs in which plasmid DNA was recognized (Fig. 3). The b– galactosidase activity per milligram of tissue protein of lung and liver showed the highest levels among organs of adult mice. No reporter gene activity was detected in any other organs which
TABLE 1
Schematic Summary of Distribution of the Transferred Gene in Vivo Plasmid form Organ
Number of copies/cell
Open circle
Linear
Supercoil
Brain Heart Lung Liver Kidney Spleen Pancreas Stomach Colon Small intestine Testis Ovary
0 2–5 20–50 10–40 2–10 2–5 2–10 1–2 0–2 0–2 0 0
2 2 1 1 2 2 2 2 2 2 2 2
2 1 1 1 1 1 1 1 1 1 2 2
2 2 1 1 2 2 2 2 2 2 2 2
Note. Seven-week-old ICR male and female mice (n 5 12) were injected i.v. with 100 mg pCMV z SPORTbgal plasmid DNA conjugated with 500 mg DMRIE-C reagent. Two days later, several organs were obtained, and the genomic DNAs from them were subjected to Southern blot analysis for the presence of transferred gene.
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istration (Fig. 4b). Although we did not show the individual results, gene expression of the transferred gene occurred equally in every fetus recovered from a dam. These results suggest that our method can successfully transfer the administered gene into a fetus and allows the gene expression transiently. The Structural Features of the DMRIE-C Reagent
FIG. 3. Transgene expression in adult mice. Mice were studied for b– galactosidase gene expression following i.v. administration of 100 mg pCMV z SPORT-bgal palsmid DNA conjugated with DMRIE-C reagent. The b– galactosidase activity per milligram of tissue protein of several organs was demonstrated (number of animals tested in each organ 5 4).
The structural characteristics of the DMRIE-C cationic liposome were studied. Electron microscopy (EM) studies have revealed that DMRIE-C (batch HCM903) forms a particle 250-500 nm in diameter (Fig. 5a) and shows multilamellar vesicles (Fig. 5c). Notable conformational changes were not detected when the DMRIE-C reagent was conjugated with plasmid DNA (Fig. 5b). These results suggest that the DMRIE-C reagent maintains the original size and feature after being complexed with DNA. It should be emphasized that the DMRIE-C/DNA complexes form no large aggregates even in the presence of serum. In contrast,
have no transferred gene. No b– galactosidase activity was detected in any of the untreated controls. Although data are not shown, expression of the reporter gene in organs of adult mice lasted up to 12 days in lung and liver, 6 days in kidney, spleen and heart, and 4 days in stomach, colon and intestine after gene transfer using DMRIE-C reagent in vivo. To evaluate the positivity of the gene transfer in vivo, tissue sections were prepared from adult liver administered with DMRIE-C/DNA complexes and then analyzed for the gene expression by b-galactosidase histochemical staining. Despite the very low number of cells, positive signals were detected in the liver (approximately 0.1 to 2.0%), whereas no positive staining was detected in the untreated liver tissues. Characteristic features of many of these cells in the liver include polygonal cellular morphology, centrally placed uniform nuclei, and localization in the hepatic plate, elucidating them as typical hepatocytes. Although the percentage of positive cells varied, positive signals were also detected in heart, lung, spleen, kidney, pancreas, stomach, colon and intestine. Gene Expression of the Progeny We next assessed the b-galactosidase gene expression in the fetuses when DMRIE-C/DNA complexes were injected at day 11.5 p.c. The proteins and total RNAs from the fetus were extracted periodically, and subjected to b-galactosidase assay and northern blot analysis, respectively. The b-galactosidase activity was detected up to 4 days after administration (Fig. 4a). The b-gal activity of the fetus at 2 days after administration was the highest of all the samples. The b-galactosidase gene transcripts were clearly detected in RNA samples from the progeny 2 days after admin-
FIG. 4. Transgene expression in the progeny. (a) After the administration of Liposome/DNA complexes into pregnant mice at day 11.5 p.c. (n 5 4), the protein from the fetus was extracted periodically at 1, 2, 3, 4 and 6 days, and was then subjected to b– galactosidase assay. (b) Two days after the administration, total RNAs were extracted from the progeny, and were then subjected to northern blot analysis by using a 32P-labeled b– galactosidase DNA fragment as a probe. Lanes 1 and 2, fetuses that received DMRIE-C and plasmid DNA; lanes 3 and 4, untreated fetuses. As a reference, transcripts of b–actin gene were analyzed.
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FIG. 5. The structural study of the liposome/DNA complex. Negative stained electron micrographs of cationic liposomes were studied on a JEM-1010 transmission electron microscope. (a) DMRIE-C alone; (b) DMRIE-C/DNA complex at optimized ratio; (c) large magnitude of DMRIE-C particle; (d) DOGS alone; (e) DOGS/DNA complex at a ratio of 1 mg DNA to 6 nmol DOGS.
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DOGS (batch 6414) forms particle 150-300 nm in diameter (Fig. 5d) and showed a conformational changes with many aggregates when mixed with plasmid DNA (Fig. 5e). In Vivo Toxicity of Cationic Liposome-DNA Conjugates The effects of intravenous cationic liposome on maternal liver were assessed by measurement of serum GOT and GPT levels. The serum GOT and GPT levels in adult animals 2 days after a single intravenous injection of 0.5 ml PBS(2) containing 500 mg of DMRIE-C reagent and 100 mg of pCMV z SPORT-bgal plasmid DNA into day 11.5 p.c. pregnant mice were 88 6 12 IU/liter and 40 6 7 IU/liter, respectively (n 5 4). Analysis of serum enzymes before the treatment revealed that the serum GOT and GPT levels were 86 6 14 IU/liter and 34 6 7 IU/liter, respectively (n 5 4), indicating there were no significant changes before and 2 days after the administration. Histopathology analysis of organs (brain, liver, spleen, lung, kidney) from these animals showed no notable abnormalities in the early phase of postadministration (data not shown). These results of the pathological and biochemical examinations demonstrated that the administration of cationic liposome/DNA complex used in this study did not show significant toxicity to animals. DISCUSSION In this study, we evaluated cationic liposome formulations suitable for systemic in vivo gene transfer into animals. We found that a new cationic lipid, DMRIE-C reagent, resulted in successful gene delivery and expression in a variety of murine organs. Despite low efficiencies when compared to dams, we also observed the DMRIE-C/DNA complexes allowed gene transfer and expression in fetuses after i.v. injection into pregnant mice at day 11.5 p.c.. When we injected the DMRIE-C/DNA complexes at day 15.5 p.c. into pregnant animals, gene transfer was observed in the neonates (Takahama et al., in preparation). No significant biochemical abnormalities or organ toxicities were observed following intravenous injection of DMRIE-C/ DNA complexes. Although we do not know the precise mechanisms of the transplacental spread of the gene, the placenta may have some important role in the delivery of biologically active factors to the developing embryos (24). This new cationic liposome formulation should provide an attractive model system to deliver biologically significant genes into animals systemically in order to look at the effect of these genes in vivo. Based on electron microscopic analysis, the DMRIE-C reagent showed multilamellar vesicles with a relatively large particle size of 250-500 nm in diameter. Although the sort of vesicles most suitable for in vivo
gene transfer is not well defined, our study showed that efficient systemic gene delivery was attained when cationic liposomes formed multilamellar vesicles. It is necessary to identify more effective cationic liposomes by synthesizing a large number of structurally related molecules and to monitor them in an appropriate in vivo assay system. Besides liposome formulation, another key factor for cationic lipid-based gene transfer in vivo is the conformational changes of liposome after complexed to DNA (1). Electron microscopic data show that addition of DMRIE-C to the plasmid DNA caused no greater changes of liposome in the mean particle diameter. Furthermore, it is proposed that ineffective gene transfer of cationic liposomes in vitro is brought about by the ready formation of a large aggregate among liposome/DNA complexes (25). In contrast, our results indicated that the complex of plasmid DNA and DMRIE-C formed less aggregation even in the blood when it was compared to other lipids such as DOGS. Considering that the large amounts of visible aggregates of DOGS/DNA complexes were mainly trapped in the lung of dams, which may reduce the gene transfer efficiency into fetuses, our success in systemic gene transfer with the DMRIE-C reagent may be partly due to a less aggregative nature of cationic lipid/DNA complexes. It is also possible that the liposome/DNA complex could exert a toxic effect in vivo. However, we did not observe significant toxicity when examining murine GOT and GPT levels after the administration of liposome/DNA complexes. In addition, no abnormalities in histopathology of major organs were present in the treated mice after 2 weeks. Thus, the DMRIE-C reagent did not show significant toxicity at the indicated conditions. In view of the safety concerns in fetal gene therapy (26), it is necessary to achieve an efficient gene delivery into fetuses without permanent integration of the gene into host chromosomes, which may in turn be passed on to the following generations. Our first report on the use of DOGS showed the transient expression of the introduced genes into fetuses without any integration of the genes into germ cells. Although we did not examine the germ line transmission of the animals after the administration of the DMRIE-C/DNA complexes, it is unlikely that they facilitated integration of the gene in vivo. Specific organ targeting in vivo might become a basis for an ideal gene therapy (27-30). Targeting to the appropriate organs will be achieved using surgical procedures, receptor-targeted uptake or a gene regulatory element such as a tissue-specific promoter/enhancer system. Interestingly, DMRIE-C/DNA complexes tend to accumulate especially in the liver and lung of animals. Although the reason for high accumulation of the liposome-DNA complex in specific organs is not fully understood, the lipid-to-plasmid charge ratio may cause organ trapping of the complex (11). Further studies are required to determine whether our method for
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fetal gene transfer is applicable for fetal organ-specific gene delivery and expression. Fetal gene therapy appears to be an ideal method to rescue embryonal dysfunction and early organ damage as the result of inherited genetic diseases (31). Recently, after our transplacental delivery with cationic liposome/DNA injection into the tail vein of pregnant mice (13), several in vivo attempts were made at gene transfer into fetuses. Pitt et al. have transduced recombinant retroviruses into the airways of fetal sheep in utero (32). A similar approach was used by McCry et al. to transfer adenovirus vectors carrying the human CFTR gene to the fetal ovine airways (33). In addition, retroviral infected fetal rat intestine was grafted subcutaneously into immune-deficient mice (34). As another interesting approach, intra-amniotic gene administration was also tested in an in vitro (35) and in vivo (36, 37) system by using cationic lipid mediated transfection, hemagglutinin virus of Japan (HVJ)liposome method, retroviral and adenoviral infection. Our nonviral cationic liposome transfection has certain advantages over these methods: (1) a simple and easy method for administration which does not require surgical intervention; (2) non-integrating vector system; (3) non-toxic and possibly non-immunogenic reagent. A major problem of cationic liposome is that liposome change its character from batch to batch and the reproducibility of the method became sometimes problematic. In this regards, gene transfer by the use of DMRIE-C as described here was reproducible when different batches were used and different scientists used the procedure. The method will be useful in analyzing the function of several genes which are important in embryonal development by introducing these genes at desired stages and into organs of embryogenesis. ACKNOWLEDGMENTS We thank Mr. T. Komatsu for animal care and Ms. M. Abe for technical assistance. This work was supported in part by a Grantin-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, by a Grantin-Aid for Cancer Research from the Ministry of Health and Welfare in Japan, and by the Bristol-Myers Squibb Foundation. Y. Takahama was awarded a Research Resident Fellowship from the Foundation for Promotion of Cancer Research.
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