Introduction

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Gene transfer is a potential therapy for many genetic and acquired diseases of the lung. In addition to transferring genetic information to cells for the production of recombinant protein to replace missing or defective proteins, gene therapy may be useful to bolster the function of injured cells or increase the susceptibility of cells to other pharmacologic agents. One of the many challenges to the clinical application of gene therapy is cell-type-selective transgene expression. Most viral and nonviral gene transfer strategies use promiscuous promoters, such as the cytomegalovirus (CMV) promoter, which drive transgene expression in most, if not all, cell types (1). Cell selectivity can attenuate the toxicity of cell-type-specific recombinant proteins in other cell types, particularly proteins requiring specialized processing, transport, or storage. The immunogenicity of transgene products may be reduced by restricting expression to a subpopulation of cells, thereby decreasing the total antigenic load. Newer gene therapy strategies that depend on the production of toxic metabolites or the manipulation of oncogenes might be most effective if transgene expression were targeted specifically to tumor cells.

Cell selectivity can be achieved in two ways. Cell surface receptors, such as lectins, or receptor antibodies have been used to target entry into specific cells. This approach has had limited success in nonviral gene transfer systems (4, 5). An alternative approach is the use of cell-type-specific promoters. Genes uniquely expressed by a particular cell type contain regulatory elements that may dictate the cell-type specificity of expression. Cell-specific enhancers have been demonstrated by plasmid transfection (6, 7), in transgenic mice (8), and more recently in selected viral vectors. Using the herpes simplex virus vector, for example, cell specificity of transgene expression has been shown for the preproenkephalin promoter (brain glial cells; 12), the tyrosinase promoter (melanocytes; 13), and the promoter from carcinoembryonic antigen (hepatocytes; 14). A common feature of these promoters is that they were derived from genes uniquely expressed by the targeted cell type. Although promoter cell specificity has been examined in replication-defective recombinant adenovirus (rAd), their use has been restricted to in vitro experimental systems without consideration of potential gene therapy applications.

The surfactant proteins A, B, and C are unique to lung, expressed only in type II and Clara cells (SP-A and SP-B), and the promoters of these genes contain regulatory elements that determine the hormonal inducibility and cell specificity of gene expression (15). Using rAd, the promoter of the rabbit SP-A gene was found to contain a lung cell-specific region that also confers cAMP inducibility in cultured rabbit type II cells, but not in the human lung cell lines A549 and NCI-H358 (19). In transgenic mice, the human SP-C gene (a 3.7-kb 5' flanking fragment) directs expression of reporter genes specifically to lung type II cells (20).

Lung-specific elements have been more fully characterized in the promoter of the human SP-B gene. Binding sequences for thyroid transcription factor-1 (T TF-1) and hepatocyte nuclear factor-3 (H N F-3) have been identified within the proximal SP-B promoter and their role in the cell-specific expression of SP-B has been demonstrated in vitro (15, 17, 23, 24). Venkatesh and coworkers (17) found that a ~ 1-kb segment of the SP-B 5' flanking sequence directs expression of chloramphenicol acetyltransferase in H-441 cells (human pulmonary adenocarcinoma cell line with Clara cell characteristics), but not in other cell lines (HeLa, Calu6, HepG2). However, tissue and cell selectivity of the SP-B promoter has not been examined in vivo. Potential advantages of the SP-B promoter for cell-selective transgene expression in the lung include its strength, which is comparable to the Rous sarcoma virus (RSV) promoter in vitro, its relatively small size, which can be advantageous in vector assembly, and its relevance for gene therapy of inherited deficiency of SP-B (25). However, because studies of the SP-B promoter were done using plasmid transfection of lung cell lines, it has been unclear whether this promoter would exhibit specificity for type II and/or Clara cells in vivo.

In this study, we hypothesized that the SP-B promoter would target type II and Clara cells in lung for transgene expression from an adenovirus vector. We linked the SP-B promoter to the bacterial reporter gene lacZ within replication-deficient adenovirus and determined the tissue distribution and cell type specificity of transgene expression in cultured cells, in explanted human fetal tissues, and in vivo in mice. We compared transgene expression to that of a similar adenovirus containing the nonspecific RSV promoter. Our results indicate that the SP-B promoter drives lung tissue- and cell-type-specific transgene expression in vitro and in vivo. A preliminary report has been published (26).

	Materials and Methods

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Cell lines were obtained from the American Type Culture Collection (Rockville, MD). A549 and NCI-H-441 (H-441) cells are derived from human pulmonary adenocarcinomas with type II and Clara cell characteristics, respectively (27). HeLa cells originated from human cervical carcinoma. Human fetal tissues from second trimester therapeutic abortions (20- to 24-wk gestation) were obtained from the Anatomic Gift Foundation (White Oak, FL). CD1 female retired breeder mice were purchased from Charles River Laboratories (Wilmington, MA) for in vivo studies. All protocols using human fetal tissues and animals were approved by the Committees on Human Research and Institutional Animal Care and Use Committees, respectively, at the University of Pennsylvania and the Children's Hospital of Philadelphia.

Adenovirus constructs were prepared by the Vector Core Facility at the Institute for Human Gene Therapy at the University of Pennsylvania. rAd.RSVlacZ has been previously described (2); it carries the bacterial lacZ gene driven by the Rous sarcoma virus (RSV) promoter. rAd.SPBlacZ carries the bacterial lacZ gene driven by the SP-B promoter consisting of the segment 641/+319 with respect to the transcription start site and with the translation start site at +14/+16 deleted (17). Transfection studies of H-441 cells showed this construct to be twofold more active than the 1039/+431 construct studied previously. The SP-B promoter sequence was removed from the plasmid (641/ +431CO_n) (17), containing the SP-B promoter and chloramphenicol acetyltransferase gene, ligated into the EcoRV/ HindIII site of pAdlink (2) to produce pAdC_a, and the lacZ cassette from XbaI-restricted pAd.CMVgal was inserted, producing pAd.SPBlacZ.

rAd.SPBlacZ was the result of homologous recombination of pAd.SPBlacZ with ClaI-digested Ad.5sub360 genomic DNA in cotransfected 293 cells. After two rounds of plaque purification, viral isolates were screened by Southern blotting to confirm promoter-transgene orientation. Virus was prepared according to standard protocol and stored at 70°C in 10% glycerol (2). Particle number was determined by measuring the A₂₆₀ of purified virus (1 A₂₆₀ unit = 10¹² viral particles) and virus was titered for plaque-forming units (pfu) on 293 cells. The rAd.SPBlacZ titer was 25 particles/pfu and the rAd.RSVlacZ titer was 55 particles/pfu for most experiments. In tail vein injection and some intratracheal instillation experiments, rAd.SPBlacZ and rAd.RSVlacZ titers were 300 and 33 particles/pfu, respectively. rAd.RSVlacZ was also titered in 293 cells for lacZ-forming units (lfu), which was 22 particles/lfu for all preparations used. This was not done for rAd.SPBlacZ because it should not express in 293 cells. Virus dose is expressed as particles/cell, particles/ml or particles/animal with particle number determined by A₂₆₀. Using this definition the multiplicity of infection (MOI) is higher than in studies that express particle number as a functional unit (plaque-forming unit or transgene-expressing unit).

Explant Culture

Human fetal tissues were carefully dissected to separate the lungs, trachea, heart, esophagus, and liver. Solid tissues were chopped into ~ 1-mm³ pieces and tubular tissues (trachea and esophagus) were sliced into rings with a McIlwain chopper as previously described (28). Explants were dispersed on 35-mm tissue culture dishes and incubated on a rocker platform in serum-free Waymouth's medium in an atmosphere of 95% air and 5% CO₂. Explants were treated with 10¹¹ viral particles in 1 ml of medium for 24 h, rinsed, and incubated for an additional 48 h in fresh medium. All assays were conducted 72 h after virus addition.

Primary Cell Cultures

Mixed cell and purified fibroblast cultures were generated from uninfected human fetal lung explants cultured for 4 d in the presence of 10 nM dexamethasone, 0.1 mM cAMP, and 0.1 mM isobutylmethylxanthine, which has previously been shown to maximally induce the differentiation of airspace-lining cells into type II cells (28). Explants were digested with trypsin, DNase, and EDTA to produce single-cell suspensions of a mixed population of cells as described previously (29). Mixed cells were plated overnight at a density of 10⁶ cells/35-mm dish for use in mixed cell cultures. In addition, some mixed cell suspension was plated for 1 h, rinsed free of unattached cells, and the attached cells cultured for four passages to yield > 99% pure fibroblast cultures.

Cell Culture

A549, HeLa, fibroblast, and mixed cells were grown in minimal essential medium and H-441 cells were grown in RPMI. All cells were grown with 10% fetal calf serum and penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (Fungizone; 2.5 µg/ml). For studies with rAd, cells were plated for 24 h and cell number was then determined from duplicate dishes by trypsinizing and counting immediately before infection. Virus dose was calculated to deliver 10³ virus particles per cell. Cells were exposed to virus for 24 h, rinsed, and incubated for an additional 48 h in fresh medium. All assays were conducted 72 h postinfection.

Animal Studies

Mice weighing approximately 30-40 g were immobilized in a body apparatus, the tails were prepared with alcohol, and virus (2.5 × 10¹¹ particles in 50 µl) was injected into a dorsal tail vein (30). Alternatively, animals were anesthetized with isoflurane and immobilized in an apparatus described previously for intratracheal instillations (31). With the animal spontaneously breathing, the trachea was visualized using a laryngoscope, intubated with a gel-loading tip, and instilled with virus (2.5 × 10¹¹ particles) in a total volume of 50 µl. To promote better distal distribution of virus (32), some animals received the perfluorochemical perflubron (LiquiVent; Alliance Pharmaceutical Corp., San Diego, CA) in aliquots of 100 µl after virus instillation until perfluorochemical could be seen freely refluxing from the trachea (total, 500-1,000 µl). All animals were sacrificed by cervical dislocation 3 d later and tissues were either stained with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) or frozen for -galactosidase assay.

X-Gal Staining

The X-Gal staining procedure included prefixing in 0.5% glutaraldehyde, washing in phosphate-buffered saline (PBS) with 1 mM MgCl₂, incubating in X-Gal solution (50 mM potassium ferricyanide, 50 mM potassium ferrocyanide, 1 mM MgCl₂, and 1 mg/ml X-Gal in PBS) at 37°C, and postfixing in formalin. Cells were incubated for 1 h in X-Gal solution, rinsed, and stored in formalin. Ex vivo-treated tissues were X-Gal stained en bloc. Lungs treated in vivo with virus in saline were inflated to 30 cm H₂O pressure with O.C.T. compound (Miles, Inc., Elkhart, IN) and PBS (1:1) before X-Gal staining en bloc. To improve distal staining of alveolar cells, lungs instilled with virus and perfluorochemical were instilled with X-Gal solution at 30 cm H₂O pressure. All tissues were incubated for 3-4 h in X-Gal solution to minimize background staining. Tissues were paraffin embedded, sectioned, and counterstained with neutral red or prepared for immunohistochemistry.

Immunohistochemistry of SP-B

We used X-Gal-stained lung tissue for colocalization studies of -galactosidase and SP-B in small airways. Slides from en bloc X-Gal-stained lung were deparaffined and permeabilized in a series of graded alcohols. Nonspecific staining was inhibited by preincubation of slides in 1.5% normal goat serum in Tris-buffered saline (TBS). Slides were then incubated overnight at 4°C in rabbit polyclonal antihuman SP-B antibody (Dr. Michael Beers; 33) at a 1:500 dilution in 0.01 M TBS-1.5% normal goat serum or in rabbit IgG at a similar dilution. Both the antibody and IgG were preincubated against mouse spleen cells to reduce nonspecific signal. After washing, slides were incubated with biotinylated goat antirabbit IgG for 1 h at room temperature, followed by blocking endogenous peroxidase activity with 0.6% H₂O₂ in methanol. Avidin-biotin complex was prepared using a Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA) and the slides were color developed using diaminobenzidine-HCl (DAB) followed by color intensification in 0.5% CuSO₄ in 0.9% NaCl. Prior to photographing, slides were coverslipped without counterstaining to preserve both the X-Gal and DAB reaction products.

Scoring Slides

Slides from rAd.RSVlacZ- and rAd.SPBlacZ-treated animals were assigned a number in random sequence and viewed by a blinded observer (P.L.B). X-Gal-positive cells were scored as type I or type II cells for analysis of alveolar staining and as SP-B positive or negative for analysis of airway cells.

-Galactosidase Assay

The -galactosidase assay was based on the method of DeFranco and Yamamoto (34). Cell pellets or tissues were sonicated in 500 µl of water and aliquots (5-25 µl) were incubated with -galactosidase buffer (60 mM Na₂HPO₄ [pH 8.0], 1 mM MgSO₄, 10 mM KCl, 50 mM 2-mercaptoethanol) with 2.2 mM o-nitrophenyl--D-galactoside (Sigma Chemical, St. Louis, MO) at 25°C. To control for endogenous A₄₂₀, a second aliquot from each tissue sample was incubated separately without substrate. To improve the sensitivity of assays done on mouse trachea with low -galactosidase activities, samples were also assayed at 37°C. When a yellow color developed, the reaction was stopped with 1 M Na₂CO₃ and the times noted for each sample. The A₄₂₀ was determined, the buffer-only control was subtracted, and the -galactosidase activity was then calculated as units of A₄₂₀/min/g protein minus the buffer-only control. Total protein was determined by the Bradford assay (35).

Statistical Analysis

Quantitative data are represented as mean ± SE and, in most cases, comparisons were made using a two-tailed t test, assuming equal variance unless otherwise indicated.

	Results

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In Vitro

We examined the activity of the SP-B promoter in rAd in human epithelial cell lines from lung and nonlung carcinomas. As a control in these and all other experiments, we used an analogous vector containing the RSV promoter, which has no known cell specificity and whose strength is similar to that of the 1039/+431 SP-B promoter in plasmid transfections of H-441 cells (17). A549 and H-441 cells appear to be derived from type II and Clara cells, respectively. A549 cells do not express endogenous SP-B by Northern and Western blot analyses; however, we have found expression of transfected SP-B promoter (641/+431) CAT reporter plasmids in these cells (unpublished data, 1996). H-441 cells express a low, basal level of endogenous SP-B mRNA, which is increased with glucocorticoid treatment (36). HeLa cells originated from a cervical carcinoma and express neither endogenous SP-B nor transfected SP-B promoter plasmids (17).

All three cell lines stained positive with X-Gal after rAd.RSVlacZ exposure (Figure 1, right) with varying levels of positive cells presumably reflecting the relative efficiency of adenovirus for infection of the cell lines. Using 10³ particles/cell of rAd.RSVlacZ, > 85% of A549 and HeLa cells were positive as compared to approximately 20% of H-441 cells. In contrast, the SP-B promoter resulted in lacZ expression in the two lung cell lines (25-30% X-Gal-positive cells) with rare expression in HeLa cells (< 1%).

View larger version (131K): [in this window] [in a new window]   Figure 1.   Cell line specificity of rAd.SPBlacZ expression assessed by X-Gal staining. Cell monolayers were exposed to 103 particles/cell of rAd.SPBlacZ or rAd.RSVlacZ, or to no virus (control), for 24 h and were X-Gal stained at 72 h. Positive X-Gal staining, typically blue, appears black in these photomicrographs. There were many X-Gal-positive A549 and H-441 cells with rAd.SPBlacZ infection, but only rare positive HeLa cells, whereas rAd.RSVlacZ infection resulted in many positive A549, H-441, and HeLa cells. Magnification is approximately ×290.

These results were corroborated by quantitative -galactosidase assay (Table 1). With rAd.RSVlacZ, -galactosidase levels for all three cell lines were 400-500 times greater than in control (uninfected) cells. -Galactosidase activity from the SP-B promoter was 60-200 times control in the lung cell lines but only 3 times control in HeLa cells. The calculated ratio for SP-B/RSV promoter activity was much lower for HeLa cells (< 0.01) than for the lung cell lines (0.37 and 0.16).

We next examined the specificity of SP-B promoter expression in primary cell cultures prepared from explanted human fetal lungs. We have previously shown that human fetal lung explants and cells cultured from the explants are susceptible to rAd infection (3). Transgene expression was measured in mixed cell cultures containing lung epithelial cells (type II cells) and fibroblasts, and in pure cultures of fibroblasts. X-Gal staining of cultures treated with rAd.-RSVlacZ showed that both fibroblasts and epithelial cells were readily infected and expressed lacZ, whereas rAd.-SPBlacZ produced staining only in the epithelial cells (Figure 2). In dose-response experiments comparing transduction in primary fibroblasts and A549 cells (Figure 3), -galactosidase activity increased in a dose-dependent manner in both fibroblasts and A549 cells infected with rAd.-RSVlacZ (10³ to 3 × 10⁴ particles/cell). rAd.SPBlacZ produced a similar dose-dependent relationship in A549 cells; however, -galactosidase activity remained consistently low in fibroblasts and did not show a dose-dependent response.

View larger version (77K): [in this window] [in a new window]   Figure 2.   rAd.SPBlacZ expression in primary cultures of human fetal lung cells assayed by X-Gal staining. Mixtures of fibroblasts and epithelial cells or purified fibroblasts from 22-wk human fetal lung explants were exposed to 103 particles/cell of rAd.SPBlacZ or rAd.RSVlacZ, or to no virus (control), for 24 h and stained with X-Gal at 72 h. Epithelial cells stained X-Gal positive with rAd.SPBlacZ infection and both fibroblasts and epithelial cells stained X-Gal positive with rAd.RSVlacZ infection. Magnification is approximately ×250.

View larger version (11K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   Figure 3.   Dose response of -galactosidase activity after infection of cultured cells. Primary human fetal lung fibroblasts and A549 cells were exposed to virus for 24 h and assayed for -galactosidase activity at 72 h. Data are the mean ± SE of triplicate dishes from a representative experiment. Error bars not visible fall within the symbol. -Galactosidase activity from rAd.SPBlacZ was dose dependent in A549 cells but not in lung fibroblasts.

Ex Vivo

SP-B promoter specificity was further examined by transducing a variety of human fetal tissues in explant culture, which exposes only surface cells to virus (3). All tissues were transduced by rAd.RSVlacZ (Table 2), with higher -galactosidase activity in lung, trachea, and heart compared to esophagus and liver. -Galactosidase activity from the SP-B promoter was detected in lung and trachea (24 and 5% of RSV level, respectively) and was very low (1-2% of RSV level) or undetectable in the other tissues.

The specific cell types within the lung and trachea that expressed from the SP-B promoter were defined by X-Gal staining (Figure 4). Cells lining air spaces (arrow) of fetal lung explants cultured for 5 d undergo differentiation into type II cells as previously described, but type I cells are not observed (28). rAd.RSVlacZ infection of cultured lung explants produced a nearly continuous layer of X-Gal-positive cells around the periphery of the explant, including both epithelial cells and fibroblasts. In rAd.SPBlacZ-infected lung explants, by contrast, staining was limited to epithelial cells lining presumptive air spaces at the surface of the explant. In tracheal explants, rAd.RSVlacZ infection resulted in dark, uniform X-Gal staining of all epithelial cells, whereas rAd.SPBlacZ infection resulted in low-level, diffuse staining of the tracheal epithelium that was comparable to control, uninfected tissue.

View larger version (104K): [in this window] [in a new window]   Figure 4.   X-Gal staining of human fetal tissue exposed to rAd ex vivo. Explants of human fetal lung and trachea, 22-wk gestation, were infected with 1011 particles of rAd.SPBlacZ or rAd.RSVlacZ or with no virus (control) in 1 ml of medium for 24 h and stained overnight with X-Gal after 72 h. In lung tissue, only epithelial cells surrounding presumptive air spaces (as shown by arrow) stained X-Gal positive with rAd.SPBlacZ. Magnification is approximately ×140.

In Vivo

We administered rAd constructs to mice by tail vein injection. rAd.RSVlacZ injection resulted in elevated -galactosidase activities, relative to control animals, in liver (50- to 500-fold) and spleen (8- to 13-fold), but not in lung or kidney (Table 3). -galactosidase activities in all tissues (liver, lung, kidney, and spleen) from rAd.SPBlacZ-injected mice were not different from control.

In initial experiments to examine promoter activity in the lung, we instilled virus intratracheally in a small volume of saline. -Galactosidase activities in lung tissue 3 d later were similar for the SP-B and RSV promoters (Table ). Because of the possibility of reflux with instillation, we also examined esophagus, stomach, and small intestine and found that -galactosidase activities were not different from control with both vectors ( 3.5 A₄₂₀/min/g protein). X-Gal staining of lung tissue after this method of instillation showed expression with both promoters primarily in airway epithelial cells. lacZ expression was not detected in trachea by -galactosidase assay with either virus, but with X-Gal staining occasional sections contained some positive cells with rAd.RSVlacZ but not rAd.SPBlacZ (data not presented). It is unclear whether this is due to variation in intratracheal administration of the viruses.

Immunohistochemistry for SP-B, as a marker for Clara cells, was positive in > 50% of cells lining both large and small airways with SP-B-positive Clara cells increasing to > 90% as airway diameter decreased (Figure 5). Sections stained using rabbit IgG as primary antibody were uniformly negative (data not shown). We examined lung sections from 11 animals treated with rAd.SPBlacZ and 8 treated with rAd.RSVlacZ and found frequent colocalization of X-Gal staining and SP-B immunoreactivity in airway epithelial cells using both viruses. X-Gal staining of airways was not uniform across sections, presumably reflecting distribution of virus within the lung, but most cells in positive regions were stained. Scoring of lacZ-positive airway cells by a blinded observer showed a trend toward more SP-B-negative cells in sections from rAd.RSVlacZ-treated mice (34% of 154 cells) than from animals exposed to rAd.SPBlacZ (19% of 205 cells).

View larger version (123K): [in this window] [in a new window]   Figure 5.   X-Gal staining of airway cells after intratracheal instillation of rAd in vivo. A representative section is shown of mouse lung that was instilled intratracheally with rAd.SPBlacZ suspended in saline and sequentially stained by X-Gal and for SP-B by immunohistochemistry. This small airway illustrates that Clara cells expressing SP-B constituted > 50% of airway cells (dark red-brown stain). SP-B in Clara cells tended to accumulate in the apex of cells (small arrow). X-Gal staining from rAd.SPBlacZ (blue cytoplasmic stain, large arrow) colocalizes to SP-B-positive cells. SP-B-positive type II alveolar cells can also be seen in areas surrounding this airway (asterisk). Magnification is approximately ×450.

Instillation of virus in saline produced few X-Gal-positive cells in lung parenchyma (data not presented). To evaluate alveolar cell type specificity, we instilled virus in saline followed immediately by perfluorochemical instillation (see MATERIALS AND METHODS) to enhance distribution to peripheral lung as previously described (32). Figure 6 illustrates representative photomicrographs of lung tissues exposed to rAd.RSVlacZ and rAd.SPBlacZ. X-Gal staining localizes to both type I and type II cells in alveoli exposed to rAd.RSVlacZ. By comparison, only type II cells appear X-Gal positive in alveoli exposed to rAd.SPBlacZ. X-Gal staining was scored by a blinded observer for number of X-Gal-positive cells that appeared to be type I, type II, macrophage, or unidentifiable by morphology. A cell was scored as type II if it was located at the alveolar surface (typically at the corners) and had a large cytoplasm:nucleus ratio with a large rounded nucleus. A cell was scored as type I if it was located at the alveolar surface and had a small dense nucleus, minimum cytoplasm, and long thin cytoplasmic projections. Macrophages were identified as cells with dense round nuclei found within the alveolar spaces. Cells not readily identifiable constituted < 6% of X-Gal positive cells. In X-Gal-stained sections of three rAd.RSVlacZ-treated animals containing a total of 33 to 129 X-Gal-positive cells per section, 58% were scored as type I alveolar cells (range, 53-64%) and 34% as type II cells (range, 33-35%). In tissue from rAd.SPBlacZ-treated animals containing 39 to 84 total X-Gal-positive cells per section, 6% were type I alveolar cells (range, 3-9%) compared to 90% type II cells (range, 86-94%). The difference in cellular distribution of lacZ expression for the SP-B promoter compared to the RSV promoter was significant (P < 0.001).

View larger version (68K): [in this window] [in a new window]   Figure 6.   X-Gal staining of alveolar cells after intratracheal instillation of rAd in vivo. Mice were intratracheally instilled with 2.5 × 1011 particles of rAd.SPBlacZ (left panel ) or rAd.RSVlacZ (right panel ) in saline, using perfluorochemical as a vehicle. Tissues were X-Gal stained at 72 h and counterstained with neutral red. Inset: Representative alveoli, with type II cells at the corners. Type I and II cells were lacZ positive with rAd.RSVlacZ, whereas only type II cells were lacZ positive with rAd.SPBlacZ. Magnification is approximately ×450; inset ×960.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

In this study we hypothesized that the SP-B promoter fragment 641/+319 would confer type II and Clara cell specificity of transgene expression in an adenoviral vector. The proximal region of the promoter (111 to 73) contains two TTF-1 sites and one HNF-3 site considered essential for basal promoter activity, and the distal region (439 to 331) has three TTF-1-binding sequences that function as a true enhancer (15, 17, 23, 24). Although the SP-B promoter demonstrated lung cell specificity in plasmid transfections of cell lines, it was unknown whether this specificity would extend to lung tissue and whether specificity would be retained in rAd. Using in vitro, ex vivo, and in vivo approaches, we found that rAd.SPBlacZ is expressed preferentially in lung type II and Clara cells, in contrast to generalized expression using the RSV promoter, which has no known cell specificity.

The recombinant adenovirus used in these studies has the E1-E3 region deleted, which cripples replication and was sufficiently large to accommodate the SP-B promoter and the lacZ gene. The recombinant virus retains viral map unit 0-1 which includes the left inverted terminal repeat (ITR), the encapsidation sequence, and the E1a enhancer (37, 38). Although the E1a enhancer was deleted to ensure cell selectivity in studies of the nicotinic acetylcholine receptor gene promoter (39), its presence did not appear to affect the selectivity of the SP-B promoter.

We chose the RSV promoter in the same rAd backbone for comparisons with the SP-B promoter because the two promoters were equal in strength in plasmid transfection of H-441 cells (17). However, when we compared -galactosidase activities after transduction of H-441 and A549 cells and lung and tracheal explants, levels were considerably higher with the RSV promoter. Differences in activity between the two rAd constructs may reflect the variety of cell types in explanted tissues, promoter strength in rAd versus plasmid, or viral titers in terms of lacZ-expressing units.

By comparison, we anticipated that intratracheal instillation of virus would result in greater -galactosidase activity with rAd.RSVlacZ than rAd.SPBlacZ because of the restricted cell type expression with the SP-B promoter. However, we found comparable levels of -galactosidase activity. This result may reflect distribution of saline-instilled virus primarily to airways. Because Clara cells comprise from 50 to 90% of cells lining murine airways, the majority of transduced cells in airways should express from both the RSV and SP-B promoters, resulting in similar levels of -galactosidase activity.

We observed low-level expression of -galactosidase from the SP-B promoter in two nonlung tissues treated ex vivo, but all tissues examined after intravenous treatment were negative. Expression was not expected in lung after intravenous treatment because the virus is delivered to endothelial cells and penetration of virus does not extend beyond one cell layer (3). By contrast, intravenous administration of rAd.RSVlacZ resulted in lacZ expression in liver and spleen, consistent with previous observations in vivo with this promoter (2). Leaky expression from the SP-B promoter is not dose dependent as indicated by our dose-response data in primary cultures of fibroblasts. This low-level expression could also be explained by impurities in the viral preparation or possibly by small numbers of epithelial cells passaged with primary fibroblasts after the initial cell preparation. Because vectors using the SP-B promoter would most likely be administered intratracheally, potential low-level leaky expression in other tissues should not pose a problem for pulmonary therapeutic applications.

We found that intratracheally instilled virus in saline did not reach adequate numbers of cells in lung parenchyma to examine type I versus type II cell specificity of lacZ expression in vivo. In a separate study from our laboratory using rabbits, Lisby and coworkers (32) demonstrated higher levels of -galactosidase activity in distal lung tissue and more complete X-Gal staining of alveolar wall cells using instillation of rAd suspended in saline plus perfluorochemical. Using this technique in mice, we found that instillation of perfluorochemical immediately following instillation of virus in saline enhanced delivery of virus to lung parenchyma. We examined X-Gal staining of alveolar cells in these animals, identifying type II cells by their appearance in the alveolar corners with a larger cytoplasm-to-nuclear ratio than type I cells (40). rAd.SPBlacZ was preferentially expressed in type II cells whereas rAd.RSVlacZ expression was similar in type I and type II cells.

Clara cells are more difficult to identify and their occurrence varies both between species and by location along the respiratory tract, ranging from 3% of cells lining large airways in humans to > 50% in mice (41). Clara cells are also a heterogeneous population exhibiting variation in morphology and function; however, as a group these cells all express immunoreactive SP-B (44, 45). Using SP-B immunostaining to identify Clara cells in airways, we found that the percentage of SP-B-positive airway-lining cells in mice was similar to estimates of Clara cell density by others (46). We demonstrated colocalization of X-Gal staining and SP-B in mouse trachea and in both large and small airways using both viruses. Although there was a trend toward increased numbers of X-Gal-positive, SP-B-negative cells using the RSV promoter, this approach has a number of limitations for quantitative assessment of cell type specificity. Additional studies using other approaches will be needed to confirm that the SP-B promoter expresses only in Clara cells of airways.

The SP-B promoter fragment was tested in a first generation rAd, a vector that is known to elicit an immune response and to lack persistent transgene expression in vivo (47). When virus was instilled with perfluorochemical there was evidence of an inflammatory response on gross inspection of the lungs (edema and congestion); microscopic findings of cellular infiltrate varied between sections and did not appear to correlate with the presence or absence of X-Gal staining. The data on cell targeting in this study should be applicable to later generation adenoviral vectors, which are less immunogenic, and possibly other viral vectors as well.

A major impetus to our studies of the SP-B promoter for cell-selective targeting of gene transfer is the condition of inherited deficiency of SP-B. This disease is most often due to an insertional mutation of the SP-B gene causing a frameshift that introduces a premature stop codon, resulting in complete absence of SP-B in lung tissue, alveolar lavage, and amniotic fluid from homozygous infants (25). Other mutations have been described in conjunction with this mutation, resulting in offspring that are compound heterozygotes. Regardless of genotype, affected infants present at term with severe respiratory distress syndrome and the only successful therapy to date has been lung transplantation (reviewed in Ref. 48). The heterozygous parents of these infants, presumably with 50% of the normal level of SP-B, are asymptomatic (49). These observations have been confirmed by animal studies of SP-B-deficient mice (50).

The pathophysiology of SP-B deficiency extends beyond increased alveolar surface tension to abnormalities in type II cells, which include increased numbers of multivesicular bodies, a paucity of lamellar bodies, and incomplete processing of SP-C (51, 52). It has been proposed that these alterations reflect a critical role for SP-B in lamellar body genesis (48). Exogenous surfactant containing SP-B has been unsuccessful in treating the disease, perhaps due to insufficient quantities of SP-B, the presence of surfactant inhibitors, or the cytostructural abnormalities that cause ongoing injury of the alveolar epithelium (53). Transfer and expression of the human SP-B cDNA to restore intracellular SP-B should correct type II cell abnormalities and permit secretion of lamellar bodies containing mature SP-B into the alveolar space and use of the SP-B promoter would target recombinant SP-B expression to type II cells. By contrast, gene transfer with promiscuous promoters such as the RSV and CMV promoters would lead to expression of recombinant SP-B within all infected lung cells, including those without SP-B-processing capabilities. Non-type II cells appear to be incapable of expressing mature SP-B and thus would potentially accumulate proSP-B and intermediate forms. Preliminary studies of nonpulmonary epithelial cell lines and primary fibroblasts in culture treated with an rAd carrying a CMV promoter and human SP-B cDNA indicate expression of the primary translation product but no 8-kD SP-B (unpublished data). Possible effects of precursor SP-B expression in non-type II cells are unclear at present but could include cell toxicity and enhanced immunogenicity from an increased protein load.

Both fetal and neonatal gene therapy strategies for treating inherited deficiency of SP-B may benefit from using the SP-B promoter. Diagnosis of SP-B deficiency is possible in utero for previously identified families due to the availability of the polymerase chain reaction, and fetal therapy would potentially allow prevention and/or correction of type II cell abnormalities. Gene therapy of SP-B deficiency could be feasible as early as the second trimester because both the endogenous promoter (45) and SP-B promoter-transgene constructs are active at this time. SP-B promoter selectivity could be advantageous in infants identified postnatally, targeting replacement gene to hyperplastic type II cells in already damaged lungs while avoiding potential damage to type I cells. When chronic lung disease is too severe and lung transplantation is preferable, SP-B gene therapy using the SP-B promoter may provide a bridge to lung transplantation by temporarily enhancing SP-B levels.

Gene therapy has been explored for oxidant injury in the lung and cell targeting by the SP-B promoter may be useful. The Clara cell is very susceptible to the damaging effects of oxygen stress and free radical damage (43) and type II cell hyperplasia is a common response to lung injury. Hyperoxia induces expression of Cu,Zn superoxide dismutase (CuZnSOD) and MnSOD in type II cells that is associated with improved tolerance to hyperoxia (54). However, 7-14 d of oxygen exposure may be required to achieve doubling of the cellular content of these enzymes. Transfer of antioxidant genes under SP-B promoter control would target susceptible lung cells and potentially provide increased antioxidant enzyme defense within 3 d.

Adenocarcinoma of the lung may be amenable to current gene therapy approaches that use recombinant wild-type p53 to induce apoptosis (55) or express herpes virus thymidine kinase (HSVtk) followed by systemic ganciclovir treatment to kill actively dividing tumor cells expressing HSVtk (56). Adenocarcinomas represent approximately 30% of lung cancers, with 50% of adenocarcinomas having bronchioloalveolar characteristics. These tumors arise from airways and invade by direct extension, making tumor cells accessible to gene therapy by the endobronchial route. The predominant cell type often has characteristics of type II and/or Clara cells (57) and expresses SP-A and/or SP-B mRNAs (27). Interestingly, A549 cells, derived from a lung adenocarcinoma, express vigorously from rAd.SPBlacZ but do not express SP-B mRNA either constitutively or upon induction (58). Thus, many bronchioloalveolar carcinomas may be susceptible to gene therapy strategies using a surfactant protein promoter to direct and limit expression of HSVtk, p53, cytokines, or other antitumor genes.

In summary, we have shown that the SP-B promoter, specifically the segment 641/+319, is lung tissue- and cell type-specific (type II and Clara cell) when used in rAd to direct transgene expression. To our knowledge, this is the first report of cell type targeting of the lung through the use of a cell-specific promoter in a viral vector. The in vivo cell selectivity of this promoter may be useful in targeting transgene expression and reducing potential toxicity associated with gene therapy in the lung. Our findings support the feasibility of developing other tissue- and cell-specific promoters for use in gene therapy.