Efficient in vivo delivery of DNA to pulmonary cells using the novel lipid EDMPC

Gene Therapy (1997) 4, 983–992 1997 Stockton Press All rights reserved 0969-7128/97 $12.00

Efficient in vivo delivery of DNA to pulmonary cellsusing the novel lipid EDMPC

CM Gorman1, M Aikawa1, B Fox1, E Fox1, C Lapuz1, B Michaud1, H Nguyen1, E Roche1, T Sawa2

and JP Wiener-Kronish2

1Megabios Corporation, Burlingame, CA, USA; and 2Department of Anesthesia and Critical Care, University of California, SanFrancisco, CA, USA

We compared the efficacy of gene transfer in vitro and in CFTR to mice and rats. We observed a lack of positive corre-vivo using various formulations of DNA–lipid complexes lation between those DNA–EDMPC formulations that deliv-based on the novel cationic lipid EDMPC (1,2-dimyristoyl- ered DNA most efficiently in vitro and those that worked bestsn-glycero-3-ethylphosphocholine, chloride salt). In vitro in vivo. Intralobar DNA delivery to rodents mediated bystudies analyzed delivery of marker genes to four estab- EDMPC was efficient. The high level of gene delivery bylished cell lines, including two of pulmonary origin. The in DNA–EDMPC formulations demonstrates that efficient lipid-vivo analysis used intralobar delivery of marker genes and mediated gene transfer to the lung is possible.

Keywords: intralobal delivery; cationic lipid; CFTR; non-viral vector; EDMPC

involved in DNA delivery to cells. In addition, theIntroductionmethod of formulation of the lipid–DNA complex, parti-

We favor the idea that safe and simple methods can be cularly the DNA to lipid ratio, is critical, since it will con-found to deliver DNA efficiently to target cells of interest tribute to the particle size and surface charge of the com-for the purpose of gene therapy. To minimize biological plex.4 Furthermore, although lipid–DNA complexes arehazards and to facilitate mass production of reagents, most often tested in vitro for convenience, the differencenonviral methods of delivery are attractive. Among the between tissue culture conditions and in vivo conditionsmethods currently under intense development is the use raises the question of whether the same formulations willof cationic lipids complexed to DNA to mediate intra- be optimal in both situations.cellular delivery of genes of interest. This method holds We have addressed these issues by studying a novelpromise for gene therapy of cystic fibrosis, because it may cationic lipid, EDMPC (1,2-dimyristoyl-sn-glycero-3-allow sufficient delivery of DNA carrying the wild-type ethylphosphocholine, chloride salt), that appeared tocystic fibrosis transmembrane conductance regulator have an enhanced ability to deliver DNA to pulmonary(CFTR) gene1 to the lung epithelium to ameliorate the tissues. To address systematically the question ofsymptoms of the disease.2,3

whether there is a correlation between the formulationsThe mechanism whereby cationic lipids facilitate DNA of lipid–DNA complexes that transfect well in vitro and

delivery is not fully understood. The lipid appears to those which lead to successful gene delivery in vivo, weinduce condensation of the DNA through charge interac- conducted a series of experiments comparing manytions, followed by interaction with the cell membrane, DNA–lipid formulations made with EDMPC. In vitroleading to endocytosis. Intrinsic fusigenic activity of the studies were carried out in a variety of established celllipid may lead to release of DNA into the cytoplasm, with lines, including two of pulmonary origin. A correspond-a portion of the DNA arriving in the nucleus.4 Many ing in vivo analysis was done using intralobar delivery ofchemically different lipids with significant ability to the complexes to the lungs of mice and rats. The resultsdeliver DNA to cells in vitro and in vivo have been indicate that the formulations that were optimal in vitroreported.4 Such lipids have been used in in vivo studies, differed from those that worked best in vivo. However,including those directed at treatment of melanoma by efficient delivery was obtained in both cases.direct injection5 and of cystic fibrosis by intralobar instil-lation or aerosol delivery.2,3 While these studies showeddelivery of DNA to the intended tissue, increased Resultsefficiency of delivery was clearly desirable. Increasedefficiency could result from chemically different lipids In vitro expression of reporter genespossessing increased ability to perform the steps The cationic lipid EDMPC was mixed in a 1:1 molar ratio

with a neutral lipid, either cholesterol or 1,2-dioleoylphosphatidylethanolamine (DOPE). To test systemati-cally the efficiency of EDMPC, DNA at different concen-Correspondence: Dr CM Gorman, 124 Belvedere Street, San Francisco,trations was mixed with the lipids in varying ratios. ACA 94117, USA

Received 31 October 1996; accepted 14 April 1997 sample of each DNA–lipid complex was retained for

Efficient delivery with DNA–EDMPC complexesCM Gorman et al

984Table 1 Physical evaluation of DNA–EDMPC formulations

Cationic Neutral lipid DNA: cationic (DNA) (mg/ml) OD at 400 nm Size (nm)lipid lipid ratio

(mg:mm)

EDMPC cholesterol 1:1 1.5 0.31 2501:1 2 0.38 2502:1 1 0.02 1622:1 1.5 0.06 1202:1 2 0.06 1583:1 0.625 0.007 157

EDMPC DOPE 1:1 1 0.23 2931:1 1.5 0.31 2502:1 1 0.17 3112:1 1.5 0.20 2502:1 2 0.19 2333:1 0.625 0.005 107

evaluation by size and optical density. These values are COS cells. The first set of DNA–lipid complexes testedincluded formulations of EDMPC–cholesterol orgiven in Table 1. DNA concentration was measured by

optical density readings taken at 260 nm. Our nomencla- EDMPC–DOPE containing equal parts of DNA and lipid(1:1) and those with a 2:1 or 3:1 excess of DNA (Figureture denotes the DNA to cationic lipid ratio (mg for DNA

and mm for the lipid) followed by the DNA concentration 2a). The formulations that transfected the least efficientlyin COS cells were those with a 3:1 ratio of DNA to lipid,in mg/ml.

The efficacy of each formulation was tested in vitro in with a DNA concentration of 0.625 mg/ml. The mostefficient formulations were EDMPC–DOPE at a 1:1 DNAseveral cell types. Cell lines such as COS and 293, which

are commonly used in gene transfer assays and in recom- to lipid ratio at 1.5 mg/ml DNA and a 2:1 ratio at 2mg/ml DNA. Frequently the EDMPC–DOPE 1:1; 1binant protein production, were tested as were estab-

lished cell lines of pulmonary origin. Expression of a mg/ml DNA complexes precipitated. During the trans-fection, EDMPC–cholesterol at 1:1; 1.5 mg/ml DNA andreporter gene transfected into these cell lines was com-

pared with expression in two lung cell lines that may be 1:1; 2 mg/ml DNA also often precipitated upon additionto the media. In general, complexes that precipitatedmore relevant model systems for cystic fibrosis. The first

series of experiments used the E. coli lacZ gene on plas- were quite efficient, suggesting a role for phagocytosis inthe mechanism of uptake in vitro.mid pMB10 (Figure 1a) as a reporter in transfections of

Figure 1 Vectors. Diagram of the expression vectors for (a) lacZ; (b) CAT; and (c) CFTR. All three vectors use the human cytomegalovirus enhancerand promoter to direct transcription of the assayed gene. All vectors also use an intron placed 5′ of the coding region to be expressed to enhance geneexpression and terminate transcription with the polyadenylation sequence from SV40. The lacZ and CAT vectors were constructed with the ampicillinresistance (ampr) marker, while the CFTR vector contained the tetracycline resistance (tetr) marker.


985diameter in the range of 200–290 nm. The 1:10 formu-lation yielded the best expression levels, up to 6 mU ofb-galactosidase activity/mg of protein. This formulationtended to form precipitates once added to the cells, againsuggesting that efficient complexes in vitro could be pre-cipitate in nature.

The above in vitro studies used COS and 293 cellswhich are commonly used for transfection studies. Sincecells of pulmonary origin could behave differently andmay be more relevant models for gene therapy of cysticfibrosis, the transfection experiments were repeated withtwo cell lines, A549 and H441, derived from lung tumors.A549 and H441 cells were transfected with complexesmade with pMB10 and EDMPC–cholesterol or EDMPC–DOPE. The complexes tested included formulations withequal parts of DNA and lipid (1:1) and formulations con-taining an excess of DNA (2:1 and 3:1). These results areshown in Figure 3a and b. While there is some variationin efficiency of these complexes, the overall pattern fortransfection with lung cells was similar to that seen withthe COS cells. Formulations made with a DNA to lipidratio of 3:1; 0.625 mg/ml transfected the least well, while

Figure 2 In vitro transfection of COS and 293 cells. Each formulationwas tested in duplicate; the standard deviation is shown by the error bars.(a) pMB10 plasmid DNA was delivered to COS cells using different ratiosof EDMPC:cholesterol and EDMPC:DOPE. Expression of b-galactosidaseis measured in milli-units of activity normalized by the amount of totalprotein in each sample. (b) p4119 plasmid DNA was delivered to 293cells using different ratios of EDMPC:cholesterol and EDMPC:DOPE, asabove. Cells were harvested 24 h later for CAT ELISA and protein assays.CAT expression levels are given in picograms of CAT protein normalizedby the amount of total protein in each sample. (c) pMB10 plasmid DNAwas delivered to COS cells using different ratios of EDMPC:cholesterol asabove. Expression of b-galactosidase is measured in milli-units of activitynormalized by the amount of total protein in each sample.

The same formulations were made using p4119 DNAcarrying the chloramphenicol acetyl transferase gene(CAT, Figure 1b) and tested in 293 cells. Complexes madewith p4119 DNA were divided for testing in vitro (Figure2b) and in vivo by intralobar delivery (see below). 293Cells were transfected, and an ELISA was used to deter-mine CAT expression levels. The most efficient wereEDMPC–cholesterol formulations for in vitro transfectionmade at a DNA to lipid ratio of 1:1, with 1.5 or 2 mg/mlDNA. For these two formulations up to 50 ng of CATprotein/mg of total protein was expressed. The leastefficient complexes in this in vitro analysis were the 2:1;1 mg/ml DNA and 3:1; 0.625 mg/ml DNA formulations.

Figure 3 In vitro transfection of two lung cell lines usingIn 293 cells, the EDMPC–cholesterol formulations werepMB10/EDMPC:cholesterol or EDMPC:DOPE complexes. A549 cells (a)more efficient than those prepared with DOPE. Com- and H441 cells (b) were transfected as in Figure 2. Expression of b-

plexes containing excess lipid, ranging from DNA to lipid galactosidase is measured in milli-units of activity normalized by theratios of 1:5 to 1:10 were also tested in COS cells (Figure amount of total protein in each sample. Each formulation was tested in

duplicate; the standard deviation is shown by the error bars.2c). These formulations yielded complexes with a mean


986 1:1; 1 mg/ml DNA and 1:1; 1.5 mg/ml DNA were best values of 800–900 mU CAT activity/mg of protein in thefor in vitro delivery. The formulations made with DOPE left lung. Significant CAT levels, 103 mU CATgenerally yielded higher expression. This result is con- activity/mg protein, were seen using water as the deliv-sistent with membrane fusion, in addition to phago- ery vehicle (Figure 4a). Since the DOPE formulations cancytosis, playing a role in the mechanism of uptake in be rather unstable, the remaining experiments were per-vitro.6 formed using EDMPC–cholesterol.

We analyzed formulations made with excess EDMPC–Expression of reporter genes in rodent lungs cholesterol in rats and found that formulations made atFor the in vivo testing, the same EDMPC–cholesterol and the 1:10 DNA:cationic lipid ratio were not efficient in vivo,EDMPC–DOPE formulations prepared using p4119 CAT even though they were among the best in vitro. The for-DNA that were tested for gene delivery in vitro (Figure mulation 1:10; 0.625 mg/ml DNA, which worked well in2b) were administered in vivo by intralobar delivery to vitro yielded expression levels of 0.07 mU in vivo.rodents (Figure 4). For delivery to the left lung of rats, 0.3 As further positive controls for efficient delivery,ml of DNA–lipid complex were administered, and CAT additional complexes at 3:1; 0.625 mg/ml DNA contain-activity was measured in lungs and trachea after 24 h. ing p4119 CAT DNA and EDMPC–cholesterol wereThe best in vivo expression was consistently seen with delivered by intralobar instillation to mice. CAT enzymea 3:1; 0.625 mg/ml DNA formulation, made with either assays performed on tissues from animals transfectedEDMPC–cholesterol or EDMPC–DOPE (Figure 4a). The with these complexes confirmed high levels of CAT3:1; 0.625 mg/ml formulation using EDMPC–cholesterol expression in the left lung. Expression levels in mice fol-yielded greater than 1 × 104 mU CAT activity/mg pro- lowing intralobar delivery were similar to the levels oftein. Given the specific activity of the CAT enzyme expression in rats (Figure 4b).(Promega, Madison, WI, USA), this value representedapproximately 100 ng CAT protein/mg of total protein.Formulations made with EDMPC–cholesterol at a ratio of Immunohistochemistry of CAT gene expression in2:1, with either 1 or 2 mg/ml DNA, gave expression at mouse and rat lungs

Immunohistochemistry was performed for detection ofCAT in mouse and rat lung. Mice were dosed by intralo-bar instillation of 100 ml of the 3:1; 0.625 mg/ml EDMPC–cholesterol formulation made with p4119 and rats weredosed with 300 ml. Tissues were harvested and preparedfor cryosectioning 48 h after treatment. Figure 5 showsimmunohistochemical detection of CAT in 4 mm thicksections of mouse lung using an anti-CAT-digoxigeninprimary antibody followed by an anti-digoxigenin gold-labeled secondary antibody and silver enhancement.Tissues at × 100 are shown in panels a–f. Sections from acontrol animal treated with 5% dextrose in water areshown in a and b. Panels d and f are bright field, whilepanels c and e are phase contrast. These are × 200 magni-fication of samples from a p4119-treated animal. In thetreated animals there were numerous fields of cells whichstained positive for CAT. In the rat, based on morphologyand double labeling of CAT expression and cell type mar-kers, the majority of cells staining positive for CAT arealveolar type I cells. Figure 6 shows double labeling ofCAT expression and type I or type II cells in 5 mm thickcryosections. CAT expression, a and b, was detected withanti-CAT digoxigenin followed by anti-digoxigeninfluorescein. Type II cells, c (arrows), were detected withanti-serum to rat surfactant protein (a kind gift from DrGurmukh Singh) followed by a rabbit 7-amino-4-methyl-cioumarin-3-acetic acid (AMCA)-labeled secondary anti-body. Type I cells, Figure 6d, were detected by the bind-ing of biotinylated Lycopersicon esculentum lectin7

Figure 4 Intralobar delivery of CAT–EDMPC complexes in rodents. followed by AMCA-labeled avidin. Corresponding phaseVarious EDMPC complexes were prepared with p4119 DNA. CAT contrast images are shown in Figure 6e and f. Because ofexpression was measured by the CAT activity assay and is given in milli-

the thickness of the cryosections, it is unclear whetherunits of activity normalized for the total amount of protein in each sample.some of the type II cells are expressing CAT or ifEach formulation was tested in triplicate; the standard deviation is shown

by the error bars. (a) Complexes were administered by intralobar delivery expression is restricted to the surrounding type I cells.(0.3 ml per rat) into the left lung. At 24 h tissues, left lung (L) and An arrow indicates one cell that did not stain positivetrachea (T), were harvested and assayed for CAT expression. (b) for CAT expression, but, is surrounded by type cells thatEDMPC:cholesterol complexes made with p4119 DNA were administered express CAT. However, no examples of individual typeby intralobar delivery into mice (100 ml per mouse). At 24 h lungs (LL,

II cells expressing CAT, without the surrounding type Ileft lobe, RL, right lobe) and trachea (T), were harvested and assayed forCAT expression. cells being positive, could be found.


987

Figure 5 Photomicrographs demonstrating CAT expression in the left lung of mice following intralobar delivery. Instillation of lipid–DNA complexesand immunohistochemistry was performed as described. (a) and (b) Control animals treated with 5% dextrose in water (×10); (c) and (d) phase andbright field, animal treated with p4119–EDMPC complexes (×10); (e) and (f) phase and bright field from an animal treated with p4119–EDMPCcomplexes (×20).

RT-PCR analysis of CFTR expression in rodent lungs mRNA-generated product (159 bp) by virtue of primerswhich span the 5′ intron present in the pMB113 vector.In order to model gene therapy experiments for treat-

ment of cystic fibrosis, we studied delivery of pMB113 When these primers were used on pMB113 plasmidDNA, a band of 501 bp was detected. The presence ofplasmid DNA carrying the CFTR gene. Following intra-

lobar delivery of 3:1; 0.625 mg/ml EDMPC–cholesterol spliced mRNA, specific for pMB113 resulted in a band of159 bp. The five mice treated with CFTR DNA were allmade with pMB113 to rodents, expression of the CFTR

cDNA was assayed at the level of mRNA. Control ani- positive for vector-specific mRNA by this analysis asindicated by the presence of a 159 bp band (Figure 7b,mals were treated with either a similar amount of 5%

dextrose (Figure 7, lane 1) or CAT expression vector lanes 3–7). This band was isolated from the gel andshown by DNA sequence analysis to be the predicted(Figure 7, lane 2). The presence of intact RNA in tissue

samples from mice was first verified by using mouse b- product of properly spliced message from pMB113.When assayed for expression of vector-specific CFTR2 microglobulin primers (Figure 7a). The samples were

then tested with vector-specific primers. The vector- mRNA, samples from both the dextrose-treated (Figure7b, lane 1) and CAT-treated (Figure 7b, lane 2) animalsspecific primers were designed to distinguish between a

vector-generated DNA PCR product (501 bp) and a were negative.


988

Figure 6 Photomicrographs demonstrating fluorescent double labeling of CAT expression and alveolar cell type markers in the left lung of rats followingintralobar delivery. Instillation of lipid–DNA complexes and immunohistochemistry was performed as described. (a) and (b) CAT expression visualizedby anti-CAT digoxigenin followed by anti-digoxigenin fluorescein. (c) The same field as in (a) showing type II cells (arrows) labeled by rabbit antiserumto rat surfactant protein followed by goat anti-AMCA. (d) The same field as in (b) showing type I cells labeled with biotinylated Lycopersicon esculentumlectin followed by avidin-D AMCA. (e) and (f) Phase contrast images of the fields in (a) and (b), respectively. Images were photographed at an originalmagnification of ×400.

has continued, it has become evident that lipid com-Discussionpounds with an increased efficiency would be of value.12

Efficient delivery of DNA to the pulmonary epithelium Some efforts have focused on the ability of these newis needed for gene therapy of diseases such as cystic lipid formulations to transfect cells in vitro.16

fibrosis. Several studies have shown in vivo delivery of In this report a direct comparison of the relationshipCFTR cDNA by adenovirus to be problematic.8–10 Thus between in vitro transfection and in vivo gene expressionalternative means of in vivo gene delivery to the nasal is described. In these studies there was a lack of positiveepithelium or lung have been developed.11–13 These correlation between the complexes which were efficientefforts using lipid–DNA-mediated gene transfer have in vitro compared with those which were efficient in vivo.succeeded in the delivery of CFTR cDNA to rodents2,3,12,13

Others have also observed that in vitro analysis for geneand have proceeded into clinical trials.14,15 As this work delivery is not predictive of expression in vivo.17–20 Even


989

Figure 7 RT-PCR detection of CFTR following intralobar delivery in mice. DNA–lipid complexes, 3:1; 0.625 mg/ml EDMPC–cholesterol made withpMB113 for CFTR expression or 4119 for CAT expression were delivered to the left lobe of the lungs of mice as described. One mouse was treated with5% dextrose alone. Twenty-four hours later, tissues (0.2–0.5 g) were homogenized in denaturation buffer. Following denaturation and centrifugationof debris, mRNA was selected by oligo-dT-cellulose. (a) Mouse b2 microglobulin primers were used to show that RNA isolated from these samples wasintact. All samples were seen to have RNA as shown by the presence of the 340 bp band. (b) CFTR vector-specific primers which span the intron yielda 501 bp when detecting plasmid DNA and a 159 bp band when detecting correctly spliced mRNA. The sample in lane 1 was from the mouse treatedwith 5% dextrose and lane 2 contains mRNA from the CAT DNA-treated animal. The five mice (lanes 3–7) treated with CFTR DNA were all positivefor vector-specific mRNA by this analysis as indicated by the 159 bp band.

the use of cell lines of pulmonary origin did not provide particles must form prior to the addition to cells.25 It isbelieved that the various precipitation methods of genea predictive model system for in vivo use.21 It remains to

be seen whether the use of polarized airway epithelial transfer, such as DEAE-dextran and calcium phosphateco-precipitation, act through phagocytosis.cells, capable of forming intact junctions, might provide

a more reasonable in vitro screening paradigm.22 In In vivo gene transfer may take place via alternativepathways. Logan et al17 measured 200–300 pg of CATaddition to there being a lack of correlation between

efficiency of in vitro and in vivo gene transfer, there was protein/mg of lung protein following intratracheal deliv-ery using DMRIE:DOPE. Lee et al20 recently reported analso a welcome lack of correspondence between com-

pounds that were toxic in vitro and their apparent lack extensive study which tested numerous novel lipids. Thebest two formulations discussed in this work yielded 300of toxicity in vivo.17,23,24

The EDMPC complexes described in this report that ng and 1 mg of CAT protein in an entire lung. All lipidstested by Lee et al were seen to be toxic both in vitro anddelivered DNA efficiently to cells in vitro were physically

different from the complexes that were most efficient in in vivo. Water has been shown active in this gene transferassay20,24 although it may to lead to osmotic cellularvivo. Complexes that were larger and had optical den-

sities of greater than 0.1 up to 0.3 (measured at 400 nm) shock.26 Meyer et al24 showed that intratracheal deliveryof DNA was best with water alone and was not facilitatedperformed better in vitro, while smaller complexes with

optical densities of less than 0.1 were optimal in vivo. The by the use of a variety of lipids.The most efficient EDMPC formulations for in vivomean diameter of the best complexes for in vivo

expression was less than 200 nm. Conversely, the com- delivery were EDMPC–cholesterol and EDMPC–DOPE at3:1; 0.625 mg/ml DNA. These formulations yielded asplexes leading to the highest expression levels in vitro

were generally greater than 200 nm. Pulmonary cell lines much as 20 000 mU of CAT activity/mg total protein.From direct measurement of intralobar delivery with thewere studied to determine whether there was any differ-

ence between transfection of lung cell lines compared p4119 CAT plasmid, we observed expression levels in therange of 50–200 ng/mg of protein or 1.5–6 mg in totalwith cell lines used for recombinant protein production

such as COS and 293 cells. While the A549 cells trans- lung The EDMPC formulations described in this reportwere an order of magnitude higher in expression thanfected well with some of these formulations, there was

still no positive correlation between complexes that DNA in water alone. In additional studies, cationic lipo-some complexes have been shown to be stable to nebuliz-expressed best in vitro (1:1; 1 mg/ml) and those that

expressed best in vivo (3:1; 0.625 mg/ml). Complexes ation, while DNA in water was degraded by this pro-cedure (Ref. 27 and Megabios, unpublished results).were also studied that contained an excess lipid to DNA

ratio. Formulations tested for in vitro efficacy in transfect- Therefore, in the aerosol delivery envisioned for treat-ment of pulmonary disease such as cystic fibrosis geneing 293 cells included formulations with an excess of lipid

to DNA. These formulations were inefficient in in vitro therapy, DNA complexed with cationic lipids would befar superior to DNA in water.transfection, with the exception of the 1:10 formulation.

This formulation tended to precipitate and did transfect Immunohistochemistry was used to visualize trans-fected cells 48 h following intralobar delivery. No stain-cells well in vitro. In general, the complexes that were

larger performed better in vitro, and precipitated com- ing was observed in control animals instilled with 5%dextrose in water (Figure 5) or in the right lungs of ani-plexes were best. This result is similar to in vitro transfec-

tion with the calcium phosphate co-precipitation tech- mals instilled with lipid–DNA complexes. No nonspecificstaining was seen in the left lungs of lipid–DNA-treatednique; for gene transfer to be successful in vitro, visible


990 animals when irrelevant digoxigenin-labeled primary TransfectionsA cationic lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphos-antibodies were used. These results are consistent with

the idea that DNA is reaching the nuclei of transfected phocholine, chloride salt (EDMPC), manufactured bySigma (St Louis, MO, USA) for Megabios Corporationpulmonary epithelial cells. Based on morphology and

double labeling of reporter gene expression and cell type was used. Liposomes were prepared by sonication ofEDMPC and cholesterol (Sigma) or EDMPC and DOPEmarkers, the majority of expressing cells in the rat are

alveolar type I cells. Since this intralobar delivery system (Sigma) at a 1:1 molar ratio. A sample of each DNA–lipidcomplex was retained for evaluation by size and opticaldeposits the complexes into the deep lung, the primary

cells exposed to the complexes include alveolar type I density. These values are given in Table 1. Sizing wasperformed on a Nicomp 370 submicron particle sizerand type II cells and alveolar macrophages. The majority

of the cells expressing CAT levels detectable by immu- (software C370, version 12.1) (Nicomp, Santa Barbara,CA, USA). Each sample was allowed to equilibrate for 2nohistochemistry were the type I cells based on mor-

phology, they were flat and exhibited large surface area min before analysis, and sizing proceeded for 5 min foreach sample.lining the alveolar wall, and on double staining of sec-

tions with antibodies to CAT and with either type I, type Plasmid DNAs (p4119 for CAT; pMB10 for lacZ andpMB113 for CFTR) were used for complexing with theII cell markers.

Expression in vivo was also followed by detection of liposomes. Expression vector pMB10 contains the humancytomegalovirus (hCMV) promoter28 the 5′ SV40 latemessenger RNA levels for the CFTR gene. Techniques

were standardized to allow isolation of intact mRNA intron, the lacZ gene and the SV40 early region polyA29

(Figure 1a). p4119 contains the hCMV promoter28,30 thefrom mouse lungs. This step was verified with the useof b-2 microglobulin. Once samples were verified for the 5′ preproinsulin intron,31 the chloramphenicol acetyl

transferase (CAT) gene32 and the SV40 early region polypresence of intact mRNA, vector-specific primers wereused to analyze transgene expression. The set of 5′ pri- A (see Figure 1b). pMB113 contains CFTR DNA

sequences33 under control of the hCMV promoter (Figuremers, demonstrated to detect CFTR mRNA specifically,was also used successfully to monitor CFTR expression 1c). These vectors also contain ampicillin or tetracycline

resistance genes and a prokaryotic plasmid origin of rep-following lipid–DNA delivery. In the experiment shownhere, 100% of the animals treated with CFTR DNA were lication. The plasmids were purified by alkaline lysis and

phenol/chloroform extraction.positive. In general, we find that greater than 75% of alltreated animals tested for vector-specific mRNA are For in vitro transfections, cells were split into six-well

plates and 18 h later were washed with 2 ml of 1 × PBS,positive.The efficiency of EDMPC formulations in vivo is high re-fed with 1 ml of serum-free medium, and incubated

for 1 h. Complexes (200 ml) were then added and left towhen compared with the results obtained in vivo byothers.17,24 The level of expression seen with EDMPC is transfect for 4 h. These conditions were found to be the

most efficient for transfecting cells in vitro with a mini-comparable with a low level of adenovirus infection20

and similar to that seen by Lee et al.20 However, unlike mum of cell death. Cells were then washed with 2 ml 1 ×PBS and re-fed with 2 ml of regular medium. Cells werethe lipids described in this latter study, lungs treatedharvested 24 h later for b-galactosidase activity assay orwith EDMPC–DNA complexes showed a visible lack ofCAT ELISA (Boehringer Mannheim) and protein assays.inflammation upon examination at high power implyingA chromogenic assay was used to assay b-galactosidasea lack of toxicity. This high level of expression and thefor the in vitro experiments. All expression data wereability to monitor CFTR expression easily by mRNA lev-standardized per milligram of soluble protein present inels suggests that lipid–DNA formulations made witheach sample as assayed by the Bradford assay kit.EDMPC can be used for efficient gene delivery to the

lungs for the treatment of a variety of pulmonary dis-In vivo deliveryeases including cystic fibrosis.Sprague Dawley female rats with a body weight of 100g were used. For intralobar instillation into the lobar lungregion, rats were anesthetized with Metofane

Materials and methods (methoxyflurane, Mallinckrodt, Phillipsburg, NJ, USA).The rats were then placed supine at an angle of approxi-mately 30° and 300 ml of lipid–DNA complex were gently

Cell lines injected into the left main bronchus using a syringeFour cell lines were obtained from the American Type attached to an intubation needle (Perfektum stainlessCulture Collection (Rockville, MD, USA): 293, a transfor- steel animal intubation needles; PGC Scientifics, Gai-med human embryonic kidney cell line (ATCC No. 1573- thersburg, MD, USA). The animal was maintained in theCRL); COS1, a SV40 transformed African Green monkey left side down position exposed to 100% oxygen for 2–3cell line (ATCC No. 1650-CRL); A-549, a human lung car- min until fully awake.26 Rats were monitored for cough-cinoma cell line (ATCC No. 174-HTB); and NCI-H441, a ing and regurgitation. The latter never occurred. Ashuman lung papillary adenocarcinoma line (ATCC No. further proof of the lack of regurgitation, five rats10809). All cell lines were maintained in DMEM F12 received Evan’s blue solution to ascertain the location ofmedium (GIBCO-BRL, Gaithersburg, MD, USA). This instillation. The blue dye was uniformly found in onemedium was supplemented with 10% fetal bovine serum lung and never in the esophagus or stomach. Rats were(GIBCO-BRL), 100 m units penicillin-streptomycin allowed to recover for 48 h before death. Pathogen-free(GIBCO-BRL), and 2 mm glutamine (GIBCO-BRL). Cells mice, 25 g, were similarly instilled with 100 ml of lipid–were maintained at a subconfluent density by splitting DNA complexes. Control animals were instilled with 5%

dextrose in water.twice weekly.


991The dosing formulations for this study comprised tant protein7 diluted in blocking buffer. The sections werewashed then incubated with anti-digoxigenin fluoresceinDNA–lipid complexes containing either EDMPC–choles-

terol or EDMPC–DOPE liposomes and an expression vec- and AMCA-labeled goat anti-rabbit (Vector Laboratories)diluted in blocking buffer. Sections were then washedtor. DNA:cationic lipid ratios are shown in Table 1. Com-

plexes were prepared the day before administration and again and coverslipped as described above.either shipped on ice overnight to UC San Francisco forthe in vivo dosing or stored at 2–8°C and used for in vitro In vivo analysis of CFTR expression by RT-PCRexperiments at Megabios Corporation. Whenever poss- Reverse transcriptase polymerase chain reaction (RT-ible, the same batch of complexes was tested in vitro and PCR) assays on whole tissues were performed using vec-in vivo. All the complexes were prepared in a solution of tor-specific primers and CFTR-specific primers. The pri-5% dextrose. For the in vivo studies, tissues were har- mers used to check for intact mRNA were as follows: forvested 24 or 48 h after dosing. Since the complexes were b-2 microglobulin: 5′ ATG TCC TCG ATC CCA GACdelivered to the left lung, the right lung served as an GGT C 3′; 5′ ACC CTG GTC TTT CTG GTG CTT G 3′.internal negative control. CAT expression was measured The expected size of the PCR product is 340 bp. The vec-by a scintillation CAT assay34 for the following homogen- tor-specific primers used were as follows: specific for 5′ization of tissues. All expression data were standardized region of CFTR message: 5′ AGA TCG CCT GGA GACper milligram of soluble protein present in each sample GCC AT 3′, forward primer (3651–3671bp in pMB113); 5′which was determined by a Bradford protein assay kit. GCT CCT AAT GCC AAA GGA AT 3′, reverse primer

(1246–1266bp in pMB113, upstream from hCFTR ATGImmunohistochemistry site).For the visualization of gene expression in mice, patho- For RNA analysis, mice were treated with complexesgen-free 25 g mice were instilled with 100 ml of lipid– (3:1, 0.625 mg/ml) of either 4119 for CAT expression orDNA complexes made with p4119. Control mice were pMB113 for CFTR expression. The CAT-treated animalsinstilled with 100 ml of 5% dextrose in water. Forty-eight had one lung analyzed for CAT expression as a positivehours following instillation, the mice were anesthetized, control for delivery (data not shown) and the other lobethe lungs and trachea were removed en bloc, and 50% served as a negative control for CFTR RNA analysis. Forornithine carbamyl transferase (OCT, Miles, Elkhart, IN, RT-PCR, tissues were flash frozen using liquid nitrogen,USA) compound in phosphate-buffered saline was then placed on dry ice and stored at −70°C. Tissueinjected through the trachea until the lungs were samples were homogenized and mRNA isolated usingexpanded to capacity. Tissues were flash frozen, and the Stratagene (La Jolla, CA, USA) RNA isolation kit fol-stored at −80°C until sectioning. Cryosections 4 mm thick lowed by poly A RNA enrichment. Reverse transcriptasewere allowed to air dry on silylated slides and were then reactions used either random hexamers or oligo dTfixed in 100% acetone. Sections were incubated with primers.buffer containing 4% normal goat serum and 1% bovineserum albumin (BSA) to block nonspecific binding sites,then incubated with anti-CAT digoxigenin (Boehringer AcknowledgementsMannheim, Indianapolis, IN, USA) diluted in the block-

We are grateful to Dr Michele Calos for her editorial helping buffer. The sections were washed then incubated within preparing this manuscript. Lee Bussey and the Biopro-a gold-labeled secondary antibody to digoxigenincessing group at Megabios Corporation provided the(Boehringer Mannheim) diluted in blocking buffer. Afterplasmid DNAs and Daisy Leung provided the liposomes.washing again, a silver enhancement was performed withClayton Bullock helped with the RNA analysis and Kris-SilvEnhance LM reagents (Zymed, South San Francisco,tina Walsh was invaluable during the preparation of theCA, USA). For the visualization of reporter genefigures. Rabbit antiserum to rat surfactant protein was aexpression and cell type markers in rats, pathogen-freekind gift from Dr Gurmukh Singh, Professor of Pathol-100 g Sprague Dawley male rats were instilled with 300ogy, Chief of Pathology and Laboratory Medicine Ser-ml of a 1:16 dilution of the 3:1, 0.625 mg/ml EDMPC–vice, Department of Veterans Affairs, Medical Center,cholesterol formulation in 5% dextrose in water madeUniversity Drive, Pittsburgh PA 15240. The antiserumwith p4119. Forty-eight hours following instillation,reacts mainly with rat surfactant protein A. Tim Heath,tissues were prepared and sectioned as described forUniversity of Wisconsin, provided the initial batches ofmice. For the simultaneous visualization of CATEDMPC; subsequent batches were purchased fromexpression and type I cells, sections were fixed in 100%Sigma.acetone then blocked with buffer containing 3% BSA. Sec-

tions were then incubated with anti-CAT digoxigeninand biotinylated Lycopersicon esculentum lectin (Vector ReferencesLaboratories, Burlingame, CA, USA) diluted in the block-

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Efficient in vivo delivery of DNA to pulmonary cells using the novel lipid EDMPC

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