Evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector

RESEARCH ARTICLE

Evaluation of a porcine model for pulmonary genetransfer using a novel synthetic vector

Steven Cunningham1{Qing-Hai Meng2{Nigel Klein3

Robin J. McAnulty4

Stephen L. Hart2*

1Respiratory Unit, Great OrmondStreet Hospital for Sick Children,Great Ormond Street, London WC1N3JH, UK2Molecular Immunology Unit,Institute of Child Health, UniversityCollege London, 30 Guilford Street,London WC1N 1EH, UK3Immunobiology Unit, Institute ofChild Health, University CollegeLondon, 30 Guilford Street, LondonWC1N 1EH, UK4Centre for CardiopulmonaryBiochemistry and RespiratoryMedicine, Royal Free and UniversityCollege Medical School, UniversityCollege London, Rayne Institute,5 University Street, LondonWC1E 6JJ, UK

*Correspondence to: S. L. Hart,Molecular Immunology Unit,Institute of Child Health, UniversityCollege London, 30 Guilford Street,London WC1N 1EH, UK.E-mail: [email protected]

{These authors contributed equallyto this paper.

Received: 5 November 2001

Revised: 30 January 2002

Accepted: 31 January 2002

Abstract

Background The pig lung, given its gross anatomical, histological andphysiological similarities to the human lung, may be useful as a large animalmodel, in addition to rodents, in which to assess the potential of vectors forpulmonary airway gene transfer. The aim of this study was to assess the utilityof the pig lung as a model of gene transfer to the human lung with a syntheticvector system.

Methods The LID vector system consists of a complex of lipofectin (L),integrin-binding peptide (I) and plasmid DNA (D). LID complexes containinga b-galactosidase reporter gene under a CMV promoter or a control plasmidat 1 mg/3 ml PBS, or 3 ml buffer, was administered to the right lower lobeof the pig lung through a bronchoscope. Pigs were culled at 48 h and lungsections prepared for immunohistochemical and histological analysis.Bronchoalveolar lavage fluid was collected and analysed for TNF-a by ELISA.

Results Immunohistochemical staining for the b-galactosidase reportergene indicated high efficiency of gene transfer by the LID vector to pigbronchial epithelium with 46% of large bronchi staining positively. There wasno evidence for vector-specific inflammation assessed by leukocytosis andcytokine production.

Conclusions This study demonstrates the use of the pig for studies of genetransfer in the lung and confirms in a second species the potential of the LIDvector for gene therapy of pulmonary diseases such as cystic fibrosis.Copyright # 2002 John Wiley & Sons, Ltd.

Keywords synthetic vectors; cystic fibrosis; gene therapy; peptide; liposome

Introduction

Gene transfer experiments in vitro and in vivo and results of clinical trialssupport the concept of treatment for cystic fibrosis (CF) by gene therapy.However, current vectors, including adenovirus, AAV and liposomes, haveachieved insufficient levels of CFTR gene transfer to bronchial epithelium inthe human lung for efficacy [1–3]. In contrast, preclinical evaluation ofadenoviral vectors, cationic liposomes and AAV in rodent models indicatedthat efficient gene transfer to bronchial epithelium was possible and that thechloride transport defect in CF mice could be corrected [4]. Rodent modelstherefore have been of great use but have failed to anticipate all of thedifficulties associated with respiratory gene transfer for CF.

The physical barriers of the murine lung, particularly the CF murine lung,differ from those found in the human lung. Human lungs possess abundantsubmucosal glands in the bronchi which mice do not, leading to the

THE JOURNAL OF GENE MEDICINEJ Gene Med 2002; 4: 438–446.Published online 12 March 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ jgm.270

Copyright # 2002 John Wiley & Sons, Ltd.

production of fewer secretions in murine lung than inhuman lung. Furthermore, CFTR mRNA levels are lowerthroughout murine pulmonary epithelium than in thehuman lung, while functional data indicates that Clx

secretion in murine lung is performed predominantly byan alternative Clx channel [5]. Murine models of CFtherefore do not develop the severe inflammatoryresponse and thickened mucus associated with CF lungdisease, which limits their value for the assessment ofefficacy of gene therapy for CF lung disease [6].Therefore, more appropriate model systems, representa-tive of the barriers to gene transfer in the human airways,are needed to develop protocols for gene therapy of CF.

Pig lungs share many anatomical and physiologicalsimilarities with those of human lungs. Their bronchishow similar patterns of branching and histology, possessa similar abundance of submucosal glands and havesimilar patterns of glycoprotein synthesis and secretion[7,8]. Similarities have also been reported in the immunesystem in the lungs of pigs and humans that may berelevant to the evaluation of inflammatory and immuneresponses to vector treatment [9,10]. The porcine lunghas already been investigated extensively as a physiolo-gical and anatomical model for human lung disease[11,12], while others have performed experimentstowards pig lung xenotransplantation in man [13,14].Moreover, pigs express CFTR in their bronchial epithe-lium and submucosal glands, which mediates secretion ofchloride and bicarbonate anions and liquid, similar tohumans [15]. Therefore, the pig is a large animal modelthat offers further opportunities to assess gene therapystrategies in the lung.

The LID vector system used in the present studyconsists of a peptide (I), with a 16-lysine (K16) DNA-binding domain and an a5b1 integrin-binding domain, acationic liposome, lipofectin (L), and plasmid DNA (D)[16]. This vector transfected rat bronchial epithelial cellsin vivo with high efficiency and with little evidence ofvector-associated inflammation [17]. The aim of thisstudy was to perform an initial assessment of theefficiency and safety of the LID vector administered tothe lungs of juvenile pigs weighing approximately 10 kg.

Methods and materials

Vector reagents

LID vector components and vector complexes wereprepared as described previously [17]. Peptide 6([K]16GACRRETAWACG) was synthesised by ZinsserAnalytic (Maidenhead, UK) and dissolved in phosphatebuffered saline (PBS). LID vector complexes containing1 mg of plasmid in a final volume of 3 ml were preparedin a weight ratio 0.75 : 4 : 1 (L/I/D) by first mixingtogether the peptide and lipid then adding the plasmidwith rapid mixing. Complexes were administered within30 min of preparation.

Plasmid pCILux was made by subcloning a luciferase

gene from pGL3-control (Life Technologies, Paisley, UK)into the eukaryotic expression vector pCI (Promega,Wisconsin, USA), while plasmid pAB11 contained ab-galactosidase reporter gene with a nuclear targetingsequence, also regulated by the CMV promoter [17].Plasmids were grown in Escherichia coli DH5a andprepared after alkaline lysis using an endotoxin-free kit(Qiagen, Crawley, UK) then dissolved in water at aconcentration of 2 mg/ml.

Piglets and bronchoscopy

Large white weaner piglets, 6 weeks of age with a meanweight of 9.8 kg, were used in this study. Animals wereaccommodated in large pens and acclimatised for 1 weekprior to study. Feed was withheld from piglets for 12 hprior to performing the bronchoscopy procedure. Pigletswere individually anaesthetised with a combination ofintramuscular Atropine (0.02 mg/kg) and Azaperon(2 mg/kg), and, once sedated, were given an intravenousdose of Propofol (3–4 mg/kg) to induce short-termsurgical anaesthesia. Animals did not require ventilationduring the anaesthesia and procedures. These procedureswere approved by the local ethics committee at theInstitute of Child Health and the Royal VeterinaryCollege, London, UK. Procedures received UK HomeOffice project license approval and followed theirguidelines.The larynx was identified by laryngoscope and then

intubated with a 4.9-mm flexible bronchoscope (OlympusXBF-P20JE; loaned by Keymed, Southend-on-Sea, UK)with a standard light source and suction. The airway wasinspected for inflammation and then the bronchoscopewas placed in the basal segment of the right lower lobe. A3-ml volume of PBS buffer or LID vector in PBS was theninstilled into the basal segment and the bronchoscopewithdrawn without suction. Animals were then allowedto recover from the procedure. The bronchoscope wassterilised using standard procedures (Cidex, Johnson &Johnson, UK) after each use. Three animals received PBS(controls), three animals LID complex with control plas-mid, and four animals were given LID complex containingpAB11.Forty-eight hours later, the animals were surgically

euthanised with pentobarbitone. Prior to death theairway was once again inspected bronchoscopically andexamined for evidence of airway inflammation, such aserythema, oedema or increased friability. The basalsegment of the right lower lobe was intubated with thebronchoscope and then lavaged with 5.0 ml of warmed0.9% sterile saline. Suction was applied to remove thelavage fluid into a trap. There was an average 44t8%recovery of bronchoalveolar lavage fluid (BALF). A guidewire was placed in the right lower lobe segment throughthe bronchoscope to ensure the correct tissue localisationat postmortem. Blood was obtained by venupunctureprior to a further lethal dose of pentobarbitone (total dose50–80 mg/kg).

Gene Transfer in the Pig Lung 439

Copyright # 2002 John Wiley & Sons, Ltd. J Gene Med 2002; 4: 438–446.

Immunohistochemistry

Pig lungs were removed immediately after euthanasia ofthe animals then transferred to the laboratory on ice anddissected within 2 h. Two representative tissue blocks(10r10r2 mm) were selected from each pig lung, oneproximal and one distal, along the bronchus of the basalsegment of the right lower lobe where the vector wasinstilled. Tissue blocks were fixed for 4 h with a mixtureof 0.2% glutaraldehyde and 2% paraformaldehyde in PBSand then embedded in OCT (Tissue Tek, Miles Labora-tories, Elkhart, IN) and stored at x70uC until sectioning.Cryostat sections of 10-mm thickness were cut andimmunostained with the avidin-biotinylated-peroxidasecomplex (ABC) method [18]. Briefly, endogenous per-oxidase was inhibited in 0.3% hydrogen peroxide and thenon-specific binding was blocked with 3% normal goatserum. Sections were immunostained with the mousemonoclonal anti-b-galactosidase (mouse IgM, Sigma, UK)overnight at 4uC. After washing in PBS, sections wereincubated with the biotinylated goat anti-mouse IgM(Vector Labs, Peterborough, UK) at room temperature for30 min and then incubated with ABC reagent (VectorLabs, Peterborough, UK) for 60 min at room temperature.Sites of peroxidase activity were visualised by developingthe slides in a solution of 3,3k-diaminobenzidine (DAB)in PBS. For antibody specificity controls, sections werestained as above with the primary antibody replaced witha non-specific mouse IgM (isotype control) and PBS(blank control). Sections were dehydrated and mountedin DPX mountant (BDH, Poole, UK) without counter-staining. They were examined and scored by microscopywith the examiner blinded to the identity of the sub-ject group. Staining was only accepted as specific tothe transfected plasmid pAB11 if the characteristicperi-nuclear staining pattern of the nuclear-targetedb-galactosidase reporter gene encoded by pAB11 wasobserved [19]. Adjacent sections from each block werestained with haematoxylin and eosin for histologicalassessment of the inflammatory response. Sectionsexamined by microscopy usually included 10–40 bronchiof different sizes and alveolar tissue was also present ineach section.

PCR detection of plasmid DNApAB11-LacZ in pig lung samples

Approximately 25 mg of bronchial epithelium wereobtained from each pig from the bronchus of the basalsegment of the right lower lobe. The epithelial sheet wasseparated in frozen tissue by peeling off from thesubmucosal tissue. Genomic DNA was extracted fromthese samples using a QIAamp DNA Mini Kit (Qiagen,West Sussex, UK). PCR was performed with 10 mg ofpurified template DNA using primers designed across thejunction of the CMV promoter and the LacZ gene withinpAB11 with the sequences 5k-AGG CGT GTA CGG TGGGAG GTC TAT-3k (forward) and 5k-CTG GCG AAA GGGGGA TGT GCT-3k (reverse). This pair of primers was used

at a concentration of 0.1 mmol/l to generate a 250 base-pair (bp) fragment in a PCR reaction buffer [16 mM(NH4)2SO4, 67 mM Tris-HCl pH 8.8 and 0.01% Tween-20; Bioline London, UK] with 0.05 units/ml Bioproenzyme (Bioline, London, UK), a thermostable DNApolymerase from Thermus flavus and 1.0 mM MgCl2 and0.1 mM dNTP (Pharmacia, St Albans, UK). The PCR cycleparameters consisted of an initial denaturation at 94uC for4 min, then 30 cycles of denaturation at 94uC for 30 s,primer annealing at 63uC for 30 s, and elongation at 72uCfor 20 s, followed by a final 1-min extension at 72uC oncompletion. The PCR products (10 ml) were electrophor-esed in 2% agarose gels containing 0.5 mg/ml of ethidiumbromide. Controls included amplification without DNAtemplate (blank control) and with DNA from cells of animmortalised human airway epithelial cell line 1HAEo-,which was used with or without being transfected withthe same vector, pAB11. PCR for the internal control,b-actin, was performed with the primers 5k-GCT GTG GCCATC TCC TGC TCG-3k (forward) and 5k-GTA TGC CTCTGG TCG TAC CAC-3k (reverse) to generate a 280-bpfragment.

Cell culture control for PCR

Human airway epithelial cells, 1HAEo- [20], were trans-fected with pAB11-b-galactosidase as a positive control toPCR of pig lung samples. The cells were maintained inEagle’s minimal essential medium (MEM; Life Tech-nologies, Paisley, UK) supplemented with 10% foetalbovine serum, penicillin (100 U/ml) and streptomycin(100 mg/ml). Cells were seeded in 24-well plates at adensity of 5r104 cells/ml for 24 h prior to transfectionthen transfected with LID vector complexes as describedpreviously [16]. DNA extractions were performed withthe QIAamp DNA Mini Kit and analysed by PCR asdescribed above.

TNF-a ELISA

Cytokine levels in bronchoalveolar lavage fluid (BALF)and in plasma were measured using a pig TNF-a ELISA kit(Endogen, Woburn, MA, USA) according to the manu-facturer’s instructions. Statistical analysis was performedusing the Student t test.

Results

Anatomical structures in pig lung

Sections were taken from either (1) the site of broncho-scopic vector instillation, or (2) a region proximal to thesite of instillation (Figure 1). Larger bronchi representingairways from the lobe to the segment and its mainbranches were identified by the presence of completecartilage rings (Figure 2a, b), whereas small bronchi,from lower down the airways, had little or no cartilage(Figure 2c). Submucosal glands were identified in large

440 S. Cunningham et al.


bronchi with a similar abundance and morphology tothose seen in human bronchi (Figure 2a).

Histochemical analysis of transfectedpig lungs

LID vector complexes containing 1 mg of plasmid DNA(pAB11 or control plasmid) or PBS alone were instilled inthe right lower lobe of pig lungs and analysed 48 h later.

There were no visible signs of airway inflammation(erythema, oedema or increased friability) either beforeor after vector administration in any of the ten pigletsexamined by bronchoscopy. In lung sections examined bylight microscopy, however, there was mild to moderatecongestion, and monocyte accumulation in some areas ofalveolar (Figure 3a, c, e) and bronchial tissues (Figure 3b,d, f), while occasional neutrophils were observed in lownumbers (<2%; not shown). Monocytes were distin-guished from neutrophils by their morphology. Histolo-gical evidence therefore suggested mild inflammation inall three experimental groups including those thatreceived LID/pAB11 (Figure 3a, b), LID/control plasmid(Figure 3c, d) or buffer alone (Figure 3e, f).

Analysis of TNF-a in BALF and serum

The cytokine TNF-a, a key mediator of the inflammatoryresponse and a marker of acute lung injury [21], wasdetected in the BALF of all three experimental groupsalthough there was no specific association with thevector-treated groups (Table 1). There was no TNF-adetected in blood plasma samples and peripheral whiteblood cell counts were all within the normal range forpigs [22] (Table 1).

The LID vector transfects pig bronchialepithelium with high efficiency

Immunohistochemical staining revealed extensive, wide-spread b-galactosidase activity in the bronchi of animalsthat received pAB11 (Figure 4a–c) whereas those thatreceived the LID complex vector control (Figure 4d) andthose that received PBS (not shown) showed nodetectable staining. Positively stained cells displayedperi-nuclear staining characteristic of nuclear-targetedb-galactosidase (nls-b-gal) [19] (Figure 4a–c). Staining inthe cells beneath the epithelium is background staining(Figure 4a) as it is highly punctuated and not nuclear-localised, in contrast to the epithelial staining. Controlsections (Figure 4d) were stained for the same time in thesame batch as nls-b-gal-transfected sections. It was notpossible to counterstain sections without masking theperi-nuclear reporter gene staining.

Figure 1. Dorsal view of pig lung showing the bronchi in thelower right lobe. Vector or buffer was administered by bron-choscope placed at position 1 within the lower right lobe.Tissue for sectioning was removed from the site of instillation(1) and from a more proximal site (2)

Figure 2. Pig bronchi of different sizes. Large bronchi (a and b) with complete cartilage rings present in the bronchial walloutside the epithelium and submucosa (solid arrows). Submucosal glands are visible in (a). (c) Small bronchi with no cartilageand alveolar tissue visible around the bronchi. Original image magnification was r100



Staining was observed in both large and small bronchi,

although at a much greater frequency in large bronchi

(Table 2). Bronchi were scored with regard to their size

and location and the number of positively transfected

cells. The proportion of cells transfected was estimated

from the area stained within bronchial epithelium in

combination with the cell number counted in adjacent

sections stained with haematoxylin and eosin in the

corresponding areas. Transfection efficiency was variable

but the bronchi themselves were scored as positively

Figure 3. Haematoxylin and eosin staining of pig lung. Alveolar (a, c, e) and bronchial (b, d, f) tissues from pigs transfectedwith plasmid pAB11 encoding a nuclear-targeted b-galactosidase reporter gene (a, b); control plasmid (c, d); or PBS (e, f).Accumulation of mild inflammatory cells was seen beneath the bronchial epithelium (arrows) and in alveolar spaces (openarrows) in all three groups. Original image magnification was r200

Table 1. Assessment of inflammatory markers. Levels of TNF-ain BALF and plasma from vector-treated and control animals.White blood cell (WBC) counts were performed in whole bloodsamples

SampleBALF TNF-a(pg/ml)

PlasmaTNF-a(pg/ml)

WBC/mm3

(normal range10 400–39,400)

PBS (n=3) 57t32 0 15 900t800Control plasmid (n=3) 73t51 (ns) 0 16 100t800LacZ plasmid (n=4) 62t57(ns) 0 14 100t900



transfected if sections contained at least 10% positive

cells, (e.g., Figure 4b). Overall, 46% of all large bronchi

stained positively while only 4.5% of smaller bronchi

were transfected (Table 2). Occasional staining of alveo-

lar cells was observed, which were carefully examined

with reference to adjacent haematoxylin- and eosin-

stained sections and identified as probable type 2 cells by

their typical morphology (Figure 4c).

Plasmid DNA is detectable in pig lungby PCR

PCR was performed on DNA extracted from lung tissue

samples located half-way between sites 1 and 2 (Figure 1)

to assess the persistent presence of plasmid DNA 48 h

after vector administration and its colocalisation with

immunohistochemical staining for b-galactosidase expres-

sion. A LacZ PCR product of 250 bp was detected in three

out of four animals in the group receiving pAB11 whereas

vector control and buffer control groups were both

negative (Figure 5). The inability to detect DNA in one of

the samples may reflect the distribution pattern of the

DNA delivered by bronchoscope within the lung. 1HAEo-

cells were transfected with approximately 40% efficiency

with pAB11 (data not shown) and used as a positive PCR

control (Figure 5).

Figure 4. Immunohistochemical staining for b-galactosidase in pig lung tissue. Animals were administered with (a)–(c)LID/pAB11 encoding b-galactosidase, or (d) LID/control plasmid. Immunoreactivity for nuclear-targeted b-galactosidase in(a) a large bronchus and (b) a smaller bronchus. (c) Probable alveolar type 2 cells (arrowed), showing characteristic peri-nuclear staining patterns (arrows). (d) Example of an area of negatively stained bronchial epithelium (arrowed) from ananimal treated with LID/control plasmid

Table 2. Transfection data from individual pigs. Three groups ofanimals were administered with LID vector containing pAB11(n=4), control plasmid (n=3) or PBS (n=3) at site 1 (distal;Figure 1) or site 2 (proximal, Figure 1). Lung sections wereimmunostained for b-galactosidase activity. Numbers signify thenumber of positively stained bronchi/total bronchi analysed

Group

Large Bronchi Small Bronchi

Distal Proximal Distal Proximal

pAB11Pig 1 2/4 2/5 1/22 2/26Pig 2 4/5 2/5 2/40 2/25Pig 3 3/5 2/6 1/38 3/31Pig 4 2/6 2/5 0/37 0/25Total 11/20 8/21 4/137 7/107Control plasmidPig 5 0/5 0/2 0/45 0/13Pig 6 0/4 0/4 0/43 0/24Pig 7 0/4 0/2 0/31 0/11Total 0/13 0/8 0/119 0/48PBSPig 8 0/4 0/2 0/39 0/29Pig 9 0/3 0/3 0/35 0/31Pig 10 0/5 0/4 0/32 0/20Total 0/12 0/9 0/106 0/80



Discussion

Shortly after the CF gene was first identified onchromosome 7 [23–25], it was shown that CFTR gene

transfer in vitro could correct the biochemical defectdemonstrating, in principle, that gene therapy for CF wasfeasible [26]. CF appeared to be a particularly promisingcandidate disease for gene therapy since it was reportedthat transfection of as few as 10% of the epithelium withCFTR may be sufficient to correct the chloride trans-port defect [27,28]. Furthermore, endogenous CFTR isexpressed at low levels in homozygous normals whileheterozygous carriers of CF mutations display normalepithelial chloride transport parameters. Despite thesefavourable factors, and the accessibility of the bronchialepithelium, clinical studies of gene therapy for CF per-

formed in both the nose and lung with a range of vectorsincluding adenovirus, cationic liposome and adeno-associated virus (AAV) have achieved insufficient levelsof gene transfer to the airway epithelium to anticipatetherapeutic efficacy [1–3].

Rodents have proven to be remarkably useful modelsfor the assessment of vector systems for gene therapyapplications, including CF. However, clinical results sug-gest that efficacy of gene transfer in humans cannot bepredicted reliably from rodent gene transfer studies. Thisprobably is due to the physiological and anatomicaldifferences between the lungs of rodents and humans.Pigs, sheep or primates may be better models for this

purpose as their lungs share similarities of size, anatomy,physiology and ultrastructure with human lung. Wepresent here the first report of respiratory gene transfer ina pig model, with the aim of assessing the potential ofthe LID vector for gene therapy of CF in terms of its safetyand efficacy.

b-Galactosidase reporter gene transfection with the LIDvector in pig bronchial epithelium was assessed byimmunostaining of lung sections for greater sensitivityand specificity in detecting reporter gene activity. Directdetection by X-gal staining was unsuccessful in thisstudy due to high levels of background staining (data notshown). Many tissue types, including lung, contain endo-genous b-galactosidase activity that contributes to non-

specific X-gal staining [29]. Therefore, immunostaining

was used since monoclonal antibodies to the bacterial

b-galactosidase do not react with endogenous mamma-

lian b-galactosidase [30]. Immunostaining for nuclear-

targeted b-galactosidase (nls-b-gal) was peri-nuclear,

consistent with previous reports that nls-b-gal associates

with the outer nuclear membrane of transfected cells and

does not enter the nucleoplasm [19]. In addition to peri-

nuclear staining of nls-b-gal, the vector control and buffer

controls were all negative for staining in all sections

examined, further reducing the possibility of false-

positive signals. The PCR data presented here indicate

that plasmid localisation is consistent with the immuno-

histological assessment of reporter gene expression

suggesting that intact plasmid DNA remains in the lung

for at least 48 h after vector administration. Experiments

using RT-PCR will be useful in future studies to quantify

gene expression.The transfection results in the pig compare well with

those from our previous study of gene transfer to the rat

lung [17]. In both species we have demonstrated efficient

transfection of bronchial surface epithelium with minimal

inflammatory response, and transfection of cells that are

probably alveolar type 2 pneumocytes, identified by

their characteristic morphology [17]. The high level of

transfection in the larger bronchi compared to the small

bronchi of the pig is likely to be a consequence of the

localised vector administration by bronchoscopy [17].

Bronchoscopic delivery aids transfection of the bronchial

epithelium and offers the advantage that the exact site of

vector administration is known for subsequent sample

analysis. However, administration of LID vector suspen-

sions by nebulisation will be important to achieve more

widespread distribution of vector and DNA throughout

the lung. In vivo transfection with nebulised polyethyle-

nimine (PEI)/plasmid DNA vector complexes achieved

widespread gene delivery and expression in the mouse

lung [31]. Nebulisation will expose the vector complex to

shearing forces. The peptide component of the LID vector

leads to increased DNA condensation compared with

lipid/DNA complexes which may offer a greater degree of

protection [16]. Indeed, nebulised polycationic com-

plexes consisting of lipid/DNA with a protamine DNA

condensing agent demonstrated greater stability and

Figure 5. Agarose gel electrophoresis of PCR. Amplified fragments of DNA from pAB11 (upper row) and b-actin (lower row)from pig bronchial epithelia. The PCR products were 250 bp for pAB11-b-galactosidase and 280 bp for b-actin. Lanes 1–10:from pig bronchial epithelia treated with control plasmid (lanes 1–3) and pAB11 (lanes 4–7) or PBS (8–10). Lane 11: cultured1HAEo- cells transfected with pAB11 (positive control). Lane 12: 1HAEo- cells untransfected. Lane 13: PCR amplification in theabsence of DNA template (no DNA control). Distinct bands for lacZ were detected in three of four pig samples from the grouptransfected with pAB11 but not in the control plasmid-treated or PBS groups



higher in vitro transfection than complexes of lipid/plasmid mixture lacking the protamine [32].

There was no evidence of vector-associated inflamma-tion in pigs receiving the LID vector at the single doseused in this study as measured by levels of TNF-a in bloodand BALF, which were the same in both buffer-treatedand vector-treated animals. In addition, white cell countsin blood were unaffected by vector administration. Thelow relative proportion of neutrophils to monocytesdetected by histological analysis of lung sections andother markers of inflammation such as alveolar thicken-ing and congestion also suggested the absence of a vector-specific inflammatory response. Previous histologicalstudies in LID-transfected rat lung at a single dosealso revealed very little evidence of inflammation [17].However, a recent analysis of vector dose response inmice demonstrated a much stronger inflammatory res-ponse in murine lungs at higher doses of vector complexthan lower doses (manuscript in preparation), indicatinga safety threshold that could be established in futureexperiments in pigs.

Gene transfer studies in pigs may be useful to augmentstudies in rodents to better assess vector efficiencyand safety. Studies using small numbers of pigs couldtherefore provide a useful bridging model betweenrodents and humans. Pig lungs better represent theobstacles to gene transfer in the human lung since theyare similar anatomically and physiologically. The dis-tribution of CFTR expression in the bronchial submucosalglands of pig and man and secretions produced are alsovery similar [15]. Inhibitors of CFTR chloride secretion inpig bronchi resulted in dehydrated, rigid mucus [33], andan accumulation of mucin and obstruction of airwaysubmucosal glands, resembling gland duct obstructionobserved in early CF disease [34,35]. The recent reportthat pigs have been cloned from somatic cells opens upthe possibility of generating genetic knockouts, and thusmodels of genetic diseases such as cystic fibrosis [36]. ACF pig would be an excellent model for the human diseasesince the similarities in anatomy of human and pig lungs,the expression pattern of CFTR in pig submucosal glandsand the effects of inhibitors of Clx secretion on mucusdeposition and rheology suggest that a CF pig woulddevelop lung disease similar to that seen in man.

In conclusion, this study supports our proposal that thepig is a practical large animal model for studies of genetherapy of pulmonary disease.

Acknowledgements

We thank Dr Linda Gibbs, Institute of Child Health, and Dr Gisli

Jenkins, UCL, for their helpful discussions. The cationic

phospholipid DOTMA/DOPE in a 1 : 1 ratio was a generous

gift from Valentis Inc. (Texas, USA). We are grateful to Luke

Bourdillon, Keymed, UK, for use of the bronchoscope. The cell

line 1HAEo- was a gift from Dieter Gruenert, University of San

Francisco, USA. We thank staff of the Royal Veterinary College

for their assistance with the animal procedures. This work was

supported by the British Lung Foundation (Grant numbers P99/

6 and P98/3).

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Evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector