Introduction

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Emphysema, a chronic pulmonary disorder distinguished by the permanent abnormal enlargement of the airways distal to the terminal bronchioles that is accompanied by the destruction of the parenchymal walls, is a leading pulmonary cause of morbidity and mortality in adults. The best-defined inherited form of emphysema is caused by a deficiency of alpha₁-antitrypsin (A₁AT), the most abundant antiprotease in human serum and pulmonary alveoli, and this condition is responsible for approximately 2% of the cases of emphysema in the United States (1). A₁AT deficiency is an an autosomal recessive disorder, with an estimated frequency of homozygotes with the most common mutation (PiZZ) of 1:6,700 in Caucasians from North America (2). Produced by hepatocytes and from mononuclear phagocytes (3), A₁AT is a glycoprotein of molecular weight 52,000 D which is secreted into the systemic circulation and diffuses into distal airways and alveoli, where it acts as the primary inhibitor of elastase released from neutrophils that are recruited in response to infectious agents or irritants (3). Decreased pulmonary levels of A₁AT result in inadequate protection of the lower respiratory tract against neutrophil elastase (NE), since the alveolar walls are vulnerable to even low levels of the protease. Destruction of lung parenchyma ensues (4, 5).

Periodic infusions with the plasma-derived human alpha₁-antitrypsin (hA₁AT), now commercially available as Prolastin, have been successful in increasing the concentration of hA₁AT in both the serum and respiratory epithelial lining fluid (ELF) to levels adequate for protection against NE (6, 7). Unfortunately, regular treatment with Prolastin, the only antiprotease currently available for use in humans, is expensive and critical shortages in its supply occur periodically. Because the protein is partially purified from pooled plasma, the possibility of blood-borne infections also exists. These issues underscore the importance of considering alternative therapeutic approaches. Because A₁AT deficiency is an autosomal recessive disorder, somatic cell gene therapy could potentially correct the defect, provided that methods of delivering normal copies of the gene to appropriate cells are safe, reliable, and efficient.

Although patients with A₁AT deficiency may develop hepatic cirrhosis due to failure to process the antiprotease (8, 9), the major clinical manifestations of this deficiency involve the lungs. It may be possible to deliver hA₁AT gene directly to the lungs rather than the liver, the predominant source of the antiprotease, in the hope that direct delivery to the site of action will reduce the total amount of protein required. Specifically, the alveoli are critical sites for correction, since the deleterious effects of NE are greatest in the lung parenchyma.

Several methods have been used to introduce the gene encoding hA₁AT into the lungs, including recombinant viruses and cationic liposomes (10). An alternative strategy, receptor-mediated gene transfer, provides specificity by exploiting the ability of receptors on the surface of cells to bind and internalize large DNA complexes that contain the gene of interest. This approach has been used to deliver functional genes to particular cell types in the lung, with one potential target being pulmonary alveolar macrophages (PAM) which reside within alveoli, interstitium, pulmonary vasculature, and airways throughout the entire respiratory tract. Investigators have shown that mannose-terminal molecular conjugates could direct the delivery of functional, exogenous genes to macrophages via the mannose receptor (11, 12). The mannose receptor is expressed by tissue macrophages, and recognizes glycoproteins with a variety of carbohydrate residues in exposed positions. Various proteins and glycoprotein conjugates bearing these carbohydrate residues bind to isolated PAM (13, 14), and mannose-terminal glycoproteins infused into the circulation of rats are eliminated from the blood by tissue macrophages (15). Such ligands could potentially target macrophages that reside in the lung. We tested this hypothesis by exploiting the mannose receptor to deliver the gene encoding hA₁AT to the lungs of rats in vivo.

	Materials and Methods

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Preparation of the Molecular Conjugate and Complexes

Molecular conjugate was constructed in which 2.0 mg of poly-L-lysine (Sigma Chemical Company, St. Louis, MO), average chain length 100 (M_r 20,000 D), was glycosylated using 89 µg of -D-mannopyranosyl phenylisothiocyanate (Sigma Chemical) dissolved in N,N-dimethylformamide (10 µg/µl concentration) (11). The solution was adjusted to pH 9.0 by the addition of 1.0 M NaCO₂, pH 9, and incubated for 16 h at 22°C. The resultant conjugate polylysine was then dialyzed against 5 mM NaCl to remove unreacted -D-mannopyranosyl phenylisothiocyanate. Approximately 0.8 to 1.0% of the -amino groups in the poly-L-lysine are glycosylated.

In these experiments, the expression plasmid pRcCMVhAAT, containing the cytomegalovirus promoter ligated to the cDNA encoding the hA₁AT, was used (16). The plasmid pGL2, containing the SV40 viral promoter and enhancer ligated to the Photonus pyralis luciferase gene and inserted into the Escherichia coli pUC19 vector, was used as an irrelevant plasmid. The plasmids were grown in DH5 strain of E. coli, extracted, and purified by standard techniques.

Plasmid DNA was dissolved in buffer containing 10 mM Tris-Cl and 1 mM ethylenediamenetetraacetic acid (EDTA), pH 8, and 5.0 M NaCl was added to yield a final concentration of 700 mM. Plasmids were condensed with mannosylated polylysine in 700 mM NaCl; the resultant charge ratio of the DNA phosphate to lysine was approximately 1:0.9. The slow addition of the polycation resulted in the formation of a turbid solution which was dissolved by the gradual, stepwise addition of 3 µl aliquots of 5.0 M NaCl. The resultant DNA complexes were examined using electron microscopy, which showed that complexes measured approximately 15-20 nm in diameter (17).

Transfection of Murine Peritoneal Macrophages in Culture

We established that macrophages isolated from rodents transfected with the plasmid pRcCMVhAAT appropriately express and secrete mature, fully processed hA₁AT. Primary macrophages were isolated from peritoneal exudates of mice 6 d after the intraperitoneal injection of 1 ml of Brewer's thioglycolate broth as previously described (11), and were maintained in tissue culture medium RPMI 1640 supplemented with 10% fetal calf serum (Gibco Laboratories, Grand Island, NY).

Approximately 10⁵-10⁶ cells were applied to 35-mm- diameter plates. Based on the cytochemical identification and morphologic characteristics, the majority of the inflammatory cells were mononuclear phagocytes. Within 24 h after isolation the cells were washed with phosphate-buffered saline (PBS), pH 7.4, and the growth medium changed. The isolated cells were treated with 2.5 µg of the plasmid pRcCMVhAAT complexed to the molecular conjugate. Cells were 40-60% confluent at the time of transfection, with a density of approximately 2 × 10⁵ cells per plate. Media pooled from six plates at various times after treatment were assayed for the production and secretion of hA₁AT. In a separate experiment, total cellular RNA was isolated from cells 2 and 8 d after transfection and analyzed for the presence of hA₁AT transcripts.

Transfection of Pulmonary Macrophages in Rats

The mannose-terminal molecular conjugate was used to transfer reporter genes into adult male Sprague-Dawley rats, each weighing approximately 250 g. In our initial experiments examining in vivo gene transfer, the conjugate was complexed to 1.0 mg of the plasmid pRcCMVhAAT and injected into three anesthetized rats. Another rat was treated with transfection complexes of the irrelevant plasmid, pGL2. Tissues (lung, liver, and spleen) from these animals were collected 4 d after treatment and analyzed for expression of the recombinant hA₁AT.

In subsequent experiments the expression plasmid pRcCMVhAAT complexed to the conjugate was injected into the tail vein in increasing doses (0.2 mg, 1.0 mg, and 2.0 mg DNA) to three cohorts of five animals each. The volumes of the solutions containing the transfection complexes ranged from 0.7 to 1.5 ml. An additional three animals were treated with 3.0 mg of the plasmid pRcCMVhAAT coupled to the conjugate. However, two of these animals injected with the highest dose of the DNA complex died 2 d after treatment and were not included in the analysis. Two additional cohorts of four rats each received transfection complexes containing either 1.0 mg of the irrelevant expression plasmid, pGL2, bound to the bona fide conjugate or 1.0 mg of the plasmid pRcCMVhAAT bound to poly-L-lysine. Five nontransfected animals were used as controls. The actual identity of the injected solutions was known to only one investigator (F.M.).

The rats were killed by carbon-dioxide narcosis 4 d after treatment. Lungs from experimental and control animals were lavaged with 5 ml of PBS, pH 7.4, and the bronchoalveolar lavage (BAL) fluid was collected. The recovered lavage fluid ranged from 3.5 to 4.0 ml. The BAL was centrifuged for 10 min at 4°C, and the supernatants were stored at 20°C. Blood (plasma) was also collected from all animals. In addition, organs were harvested from control and transfected rats. The fluid samples and tissue were analyzed for expression of the recombinant hA₁AT. The animal research protocol was reviewed and approved by the Case Western Reserve University Institutional Animal Care Committee (Cleveland, OH).

Detection of hA₁AT mRNA in the Lung

The presence of mRNA transcripts for the hA₁AT gene in the lungs of transfected animals was determined after treatment of total cellular RNA with Moloney Murine Leukemia Virus reverse transcriptase (RT) and amplification of the resultant cDNA by polymerase chain reaction (PCR). Total cellular RNA was isolated from nontransfected, mock-transfected, and transfected tissues using standard techniques. One microgram of total cellular RNA from tissues or cultured cells was treated with 10 U DNAse I (Boehringer Mannheim, Indianapolis, IN) to remove contaminating genomic or plasmid DNA from the sample. The RNA sample was heated to 80°C for 10 min, then precipitated with ethanol to remove the DNAse I. The precipitated RNA was suspended in diethylpyrocarbonate-treated water, then added to a solution containing 500 nM of the (dT)₁₆ oligonucleotide primer and 500 nM of each dNTP, and heated to 42°C for 2 min. One microliter of Superscript II Reverse Transcriptase (Gibco Laboratories) was added, and the solution was incubated for 30 min at 42°C. The reaction was terminated by heating the tubes at 90°C for 5 min. One microliter of RNAse H was then added and incubated for 20 min at 37°C.

Two microliters of the cDNA pool was amplified (Perkin Elmer Cetus, Norwalk, CT) using standard techniques. The cDNA sample was added to a solution containing 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl₂, 0.1 mg/ ml bovine serum albumin (BSA), 200 µM dATP, 200 µM dCTP, 200 µM dTTP, and 200 µM dGTP; oligonucleotide primers and Taq polymerase were added immediately before amplification. The DNA was amplified through 30 cycles, using the following primers for the hA₁AT: GTA ATC GAC AAT GCC GTC TTC TGT CTC G, a primer that binds to the 5' end of the hA₁AT cDNA corresponding to positions 5 to 32; and TTA AAC ATG CCT AAA CGC TTC ATC ATA GGG, an antisense primer that corresponds to nucleotide positions 737 and 766. The predicted length of the amplified region of DNA was 761 base pairs. In addition, the cDNA was also amplified using a sense primer outside the hA₁AT cDNA in the T7 promoter (GTA ATA CGA CTC ACT ATA GGG), and the antisense hA₁AT cDNA primer, to ensure that transgene expression in the murine macrophages was being detected. The expected length of this amplified DNA was 822 base pairs. As a control, rat glyceraldehyde-3-phosphate-dehydrogenase (rGAPDH) mRNA was also amplified using similar conditions with primer CCA TGG AGA AGG CTG GGG C, which corresponds to positions 371 to 389 in the rGAPDH cDNA; and primer CAA AGT TGT CAT GGA TGA CC, an antisense primer which corresponds to positions 546 to 565. The expected size of the amplified cDNA generated from the rGAPDH mRNA was 194 base pairs. Ten microliters of the reaction products were separated by gel electrophoresis on a 1.5% agarose gel. Southern blot hybridization was performed with radiolabeled probes using standard techniques.

In Situ Hybridization of hA₁AT mRNA

Tissues were harvested after transfection with the complexes, and in situ hybridization of hA₁AT mRNA was performed. The tissues were fixed in 2% paraformaldehyde in PBS, pH 7.4, overnight at 4°C; paraffin was embedded; and sections (5 µm) were prepared and placed on polylysine-coated slides. The sections were deparaffinized at 60°C for 10 min, cleared in xylene, and rehydrated through ethanol washes. Immediately before hybridization, the sections were treated with 1 µg/ml proteinase K in a moisture chamber for 15 min at 37°C. The slides were washed and the tissue sections were dehydrated by sequentially incubating the slides in increasing concentrations of ethanol, and then air-dried at room temperature.

Sense and antisense riboprobes containing a segment labeled with [³⁵S]UTP (DuPont-New England Nuclear, Boston, MA) were synthesized in vitro from the 3' region of the hA₁AT cDNA of the plasmid transcription vector using standard techniques. The sections were blocked and 200 µl of hybridization solution, containing 50% deionized formamide, 0.3 M NaCl, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, Denhardt's Solution, 500 µg/ml yeast tRNA, 500 µg/ml PolyA, 50 mM DTT, and 10% polyethylene glycol (MW 6,000 D) and radiolabeled riboprobes (2 million cpm per slide), was applied. The tissues were heated to 80°C for 10 min, then incubated in a moisture chamber for 4 h at 45°C. The sections were washed vigorously to remove free probe, incubated with RNAse A and RNAse T1 for 15 min at 22°C, washed again, then dehydrated with ethanol washes. The dried slides were coated with Kodak NTB-2 autoradiographic emulsion (Kodak Corporation, Rochester, NY), placed in a light-tight box, and exposed at 4°C for 14-16 d. The slides were then developed, counterstained with nuclear fast red, and mounted. Adjacent tissue sections were also stained with hematoxylin and eosin.

Enzyme-linked Immunosorbent Assay for hA₁AT

Spent culture media, plasma, and concentrated BAL fluid were analyzed for the presence of hA₁AT using an ensyme-linked immunosorbent assay (ELISA) as previously described (18). The values of the human antiprotease detected in the lung were corrected for the volume of respiratory ELF recovered by measuring urea dilution (19). The limit of detection of the hA₁AT was 0.5 pM. Significantly, the endogenous rat A₁AT was not detected by this ELISA.

Measurement of hA₁AT Functional Activity

The inhibition of NE hydrolysis of methoxy-succinyl-ala-ala-pro-val-nitroanilide (Sigma Chemical), a synthetic chromogenic substrate of NE, by human A₁AT in spent culture media from the transfected cells was measured to determine if the antiprotease secreted by the murine peritoneal macrophages was functional. The change of absorbance at 410 nm of these samples was measured using a Beckman spectrophotometer, and the relative content of active NE was determined by comparison with control incubations consisting of NA and buffer.

Western Blot Analysis for hA₁AT

Media samples (20 µl) obtained at different times after in vitro transfection of PEM were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%) and transferred onto nitrocellulose membrane filters using standard techniques. The blots were blocked with PBS, pH 7.4, 0.03% polyoxyethylenesorbitan monolaurate (Tween 20), and 10% (wt/vol) dry skim milk for 1 h at room temperature, followed by incubation with a 1:1,000 dilution of polyclonal rabbit-derived anti-hA₁AT antibody (Sigma Immunochemicals, St. Louis, MO). The membrane was washed three times in PBS, pH 7.4 and 0.03% Tween 20, then incubated with a 1:1,000 dilution of goat antirabbit IgG-horseradish peroxidase conjugate. The membrane was then washed vigorously four times with PBS, pH 7.4 and 0.03% Tween 20, and 10 ml of Western blot enhanced chemiluminescence detection solution was applied for 1 min. The luminescence emitted from the filter was detected by a 1-min exposure to photographic film.

Immunocytochemical Staining for hA₁AT in Macrophages

Individual cells in culture and tissues expressing hA₁AT were identified using immunohistochemical means. BAL fluid was centrifuged for 10 min, and the cells were applied to eight-well slides. The cells were fixed with 2% paraformaldehyde and 0.1% Triton X-100 in PBS, pH 7.4, for 20 min at 37°C, then washed repeatedly with PBS, pH 7.4, to remove the fixative. The cells were incubated sequentially with a rabbit-derived anti-hA₁AT (Sigma Chemical) and alkaline phosphatase-conjugated goat F(ab')₂ directed against rabbit IgG (Sigma Chemical). The primary and secondary antibodies were diluted 1:1,600 and 1:400, respectively, in PBS, pH 7.4. Between each incubation, the cells were washed three times with PBS, pH 7.4.

Lungs, livers, and spleens from experimental and control rats were harvested and fixed in 2% paraformaldehyde in PBS, pH 7.4, overnight at 4°C, then washed several times with PBS, pH 7.4. The tissues were embedded in paraffin, sectioned (5 µm), and treated with proteinase K for 10 min at 37°C. Tissue sections were incubated with 1% BSA in PBS, pH 7.4, then washed. The rabbit polyclonal antibody against hA₁AT diluted 1:400 in PBS, pH 7.4, was applied overnight at 4°C. As controls, neighboring sections were also incubated with the same rabbit anti-hA₁AT antibody that was adsorbed with Prolastin (Miles Incorporated, West Haven, CT). The sections were washed with PBS, pH 7.4, then incubated with a 1:200 dilution of alkaline phosphatase-conjugated goat F(ab')₂ directed against rabbit IgG in PBS, pH 7.4, for 1 h at 37°C in a moisture chamber. The sections were washed again with PBS, pH 7.4. Vector Red (Vector Laboratories, Burlingame, CA) was used as the chromagen, and this stain was applied to the BAL cells and tissue sections for 3 and 5 min, respectively. The stained sections were counterstained with hematoxylin for 2 min, mounted, and examined by light microscopy.

Identification of Alveolar Macrophages

Tissues and cells isolated from BAL were identified using immunohistochemical and cytochemical means. Tissue sections were fixed with 2% paraformaldehyde, washed with PBS, pH 7.4, then sequentially incubated with a 1:100 dilution of murine monoclonal antibody MAB1435 (Chemicon International Inc., Temecula, CA), which recognizes a single chain glycoprotein expressed on lysosomal membrane of tissue macrophages in rats, and 1:400 dilution of alkaline phosphatase-conjugated goat antibody directed against mouse IgG (Sigma Chemical). Both antibodies were diluted in PBS, pH 7.4, and between incubations the cells were washed with PBS, pH 7.4. The cells were then stained with Vector Blue (Vector Laboratories) for 3 min as described by the manufacturer. Stained cells and sections were counterstained with nuclear fast red for 1 min, mounted, and examined. Cells grown in culture were also stained for nonspecific esterase, a cytochemical marker for mononuclear phagocytes, using standard techniques (11).

Statistical Analysis

Data are expressed as the mean ± standard error of the mean (SEM). Statistical comparisons between the production and secretion of human A₁AT in the ELF from control and treatment groups were made using paired Student's t tests (20).

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Expression of hA₁AT by Murine Peritoneal Macrophages In Vitro

Initial experiments examined the transfer and expression of plasmids containing the gene encoding hA₁AT in murine peritoneal macrophages via the mannose receptor. Five micrograms of plasmid DNA were complexed to the mannose-terminal molecular conjugate and applied to cells in culture for 24 h. The expression of hA₁AT was confirmed by Northern blot hybridization of total cellular RNA isolated from the transfected macrophages. A 1.3- kb transcript, the expected size of hA₁AT mRNA, was detected in total cellular RNA 2 d after transfection (data not shown). Moreover, hA₁AT transcripts were found in transfected peritoneal macrophages by treating total cellular RNA with RT and amplifying the resultant cDNA by PCR using primers specific for the transgene. The hA₁AT mRNA was again detected in the transfected cells 2 d after transfection (Figure 1). Expression was short-lived; no transcripts were identified using either means of detection 8 d after transfection. The endogenous murine A₁AT was not detected in the RNA isolated from nontransfected and mock-transfected cells.

View larger version (41K): [in this window] [in a new window]   Figure 1.   Detection of hA1AT mRNA transcripts in transfected peritoneal macrophages. Total cellular RNA extracted from murine peritoneal macrophages in primary culture 2 and 8 d after transfection were treated with or without RT and, using specific oligonucleotide primers in the hA1AT transgene, the resultant cDNA were amplified by PCR (left panels). In addition, the same cDNA was amplified using primers complementary to sequences in the T7 promoter and hA1AT gene to establish that transcripts detected were from the exogenous gene (right panels). Total cellular RNA from nontransfected (NT) cells were used as controls. The reaction products were separated by agarose gel electrophoresis and analyzed by Southern blot hybridization using labeled cDNA probes. Molecular weight standards (base pairs) are indicated in the right margin, and a schematic diagram of the transgene and oligonucleotide primers are shown below.

Culture media from murine macrophages were collected at different times after transfection and examined for the production and secretion of hA₁AT using an ELISA. The hA₁AT was detected in spent media from transfected murine macrophages 48 and 72 h after transfection (Table 1). Western blot analysis of the culture media from the transfected cells demonstrated the presence of the mature, fully processed hA₁AT (Figure 2). Transgene expression was short-lived, and no human antiprotease was detected beyond 96 h after transfection. No A₁AT was detected in nontransfected cells or murine macrophages treated with plasmid DNA alone. This finding is significant because production of endogenous A₁AT is upregulated by lipopolysaccharide (21), a frequent contaminant of DNA preparations (22). Thus the production of the hA₁AT by transfected cells, detected by ELISA and Western blot hybridization, was concordant with transgene transcription.

View larger version (39K): [in this window] [in a new window]   Figure 2.   Detection of secreted hA1AT in culture media of transfected murine peritoneal macrophages. Spent culture media were collected from peritoneal macrophages during a 24-h period at different times after transfection and pooled for analysis. Proteins in the pooled media were separated by denaturing PAGE. Media samples (20 µl) from nontransfected cells (NT), mock transfected cells treated with plasmid DNA alone (MT), and cells 1 d, 2 d, 6 d, and 10 d after transfection were analyzed for hA1AT. Purified, pooled hA1AT was used as a positive control (P). Molecular weight standards (kD) are shown in the right margin.

Production of hA₁AT by Pulmonary Macrophages in Rats

The mannose-terminal molecular conjugate was used to transfer the gene encoding hA₁AT into the lungs of intact animals. Sprague-Dawley rats were anesthetized and underwent systemic injections of increasing doses of complexed plasmid DNA (0.2 mg, 1.0 mg, 2.0 mg, and 3.0 mg), and 4 d after treatment with transfection complexes the animals were killed and tissues examined for expression of the hA₁AT transgene. All of the rats that received the transfection complexes containing 0.2 mg, 1.0 mg, and 2.0 mg of the expression plasmid tolerated the procedure well. However, two of three animals injected with the highest dose of the DNA complex (3.0 mg) died 2 d after treatment; histopathologic analysis of the lungs revealed evidence of pulmonary hemorrhage. No inflammation was detected in lungs from nontransfected, mock-transfected, or transfected mice.

Transcripts expressed from the hA₁AT transgene in the lungs of nontransfected, mock-transfected, and transfected mice were determined by treating total cellular RNA with RT and amplifying the resultant cDNA by PCR using primers specific for the transgene (Figure 3). Human A₁AT mRNA was found in the lungs from six of the 13 rats (46%) that received the higher doses (1.0 and 2.0 mg) of plasmid DNA (Table 2). The transgene mRNA was not detected in the animals injected with the lowest dose (0.2 mg) of plasmid DNA (Table 2). The RNA samples were evaluated in the presence and absence of RT to ensure that contamination with plasmid DNA had not occurred; no signal was noted in the absence of RT. The plasmid (pRcCMVhAAT) bound to poly-L-lysine was also expressed in three of the four rats injected with these complexes (Table 2). The lungs from nontransfected rats and animals treated with complexes prepared with an irrelevant plasmid (pGL2) bound to the bona fide conjugate did not express the transgene (Table 2). The mRNA from the endogenous gene encoding rGAPDH was identified in the corresponding samples (data not shown).

View larger version (67K): [in this window] [in a new window]   Figure 3.   Expression of hA1AT mRNA transcripts in the lungs of rats following treatment with transfection complexes. Total cellular RNA was isolated from the lungs of rats 4 d after intravascular injection with complexes containing the mannosylated polylysine bound to 1.0 mg of the plasmid pRcCMVhAAT (T), polylysine bound to 1.0 mg of pRcCMVhAAT (IC), or mannosylated polylysine bound to 1.0 mg of an irrelevant plasmid, pGL (ID). Total cellular RNA from a nontransfected animal (NT ) was also used as control. The extracted RNA was treated with or without RT, and the resultant cDNA was amplified by PCR (nonquantitative) using oligonucleotide primers within hA1AT cDNA. The reaction products were separated by agarose gel electrophoresis (upper panels) and analyzed by Southern blot hybridization using radiolabeled cDNA probes (lower panels). Blots shown are from two separate experiments. Molecular weight standards (base pairs) are indicated in the right margin.

The cellular distribution of transgene expression in the lungs was established by in situ hybridization of hA₁AT mRNA. Four days after injection with transfection complexes, lung sections from rats in each treatment and control group were examined (Figure 4). Expression of hA₁AT was not uniformly distributed throughout the transfected lung. Clusters of positive cells were observed, and the signal for transgene mRNA was most prominent in cells around the vasculature in the lungs of rats that received higher doses of plasmid DNA. Not all vascular sites had cells expressing the hA₁AT transgene. Few cells located in the alveolar space expressed the hA₁AT transgene (Figure 4). No signal was detected in lungs with the sense probe. The lungs from nontransfected and mock-transfected animals did not exhibit a signal.

View larger version (128K): [in this window] [in a new window]   Figure 4.   Detection of hA1AT mRNA transcripts in pulmonary tissues. Four days after systemic injection with transfection complexes containing the plasmid pRcCMVhAAT bound to mannosylated polylysine or polylysine alone, rats were killed, and lung sections were processed and sectioned. Radiolabeled sense (right column) or antisense (middle column) riboprobes to the hA1AT transgene were applied to sections of lung parenchyma, exposed to a photographic emulsion for 14-16 d, then counterstained with nuclear fast red. Brightfield (left column) and darkfield (middle column) views of the identical region of lung from a rat that received the bona fide transfection complexes (a-c) are shown. Cells with positive signal had a perivascular distribution in the lungs. Representative lung sections from an animal that received the plasmid pRcCMVhAAT complexed to polylysine (d-f  ) or injected with complexes containing an irrelevant plasmid (g-i) are also shown. No hybridization signal was detected in these tissues. Original magnification of the photomicrographs: ×100.

The lung was examined for expression of the transgene product. The hA₁AT was detected in cells around the bronchial and small pulmonary arteries in the lungs from rats treated with transfection complexes, which corresponded to the nonhomogenous pattern of expression found by in situ hybridization of transgene mRNA. Much of the staining was localized to the perivascular regions of the lung, and rare macrophages residing in the alveolar space from transfected animals expressed hA₁AT (Figure 5). Expression of the hA₁AT transgene was not detected in alveolar pneumocytes or airway epithelium. Thus the delivery of the exogenous gene to the lung appears to be a specific process, targeting only the PAM. As described previously (11), cells in the marginal zones of the spleens from transfected rats also stained positive for the transgene product.

View larger version (126K): [in this window] [in a new window]   Figure 5.   Expression of hA1AT in rat pulmonary macrophages. Four days after intravascular injection with mannosylated polylysine complexed to the plasmid pRcCMVhAAT, lung sections underwent immunohistochemical staining for hA1AT. Sections of lung show a bronchus, bronchial artery, and surrounding parenchyma from rats treated with transfection complexes containing the expression plasmid pRcCMVhAAT after immunohistochemical staining with an anti-hA1AT (a). As a control, an adjacent section was incubated with the primary antibody adsorbed with Prolastin (b). Different lung sections show alveoli after immunohistochemical staining with an anti-hA1AT (c) and a neighboring section with antibody that has been adsorbed with Prolastin (d). Cells (red-stained) expressing hA1AT had a perivascular and alveolar distribution, and are indicated by arrows. Cells isolated from BAL (e) of a rat transfected with complexes containing 1.0 mg of the expression plasmid after immunohistochemical staining are also shown. Again, positive cells are identified by arrows. Original magnification of the photomicrographs: (a) and (b), ×100; (c), (d), and (e), ×200.

Cells isolated from the BAL were similarly examined for expression of the transgene. More than 90% of the cells extracted from the lavage fluid were PAM, based on the immunocytochemical identification and morphologic characteristics of the cells, and less than 1% of the macrophages isolated from transfected animals expressed hA₁AT (Figure 5). No cells in the lungs of nontransfected and mock-transfected animals were positive for the transgene product.

Plasma and BAL samples from rats were analyzed for production and secretion of hA₁AT using an ELISA of the hA₁AT (Figure 6). The hA₁AT was not found in the plasma and BAL from nontransfected animals and rats treated with the complexes prepared with an irrelevant plasmid (pGL2) bound to the bona fide conjugate or the expression plasmid (pRcCMVhAAT) bound to a poly- L-lysine. The levels of hA₁AT in the ELF increased in a dose-dependent fashion. The highest concentration of the human antiprotease was found in the lungs of rats treated with transfection complexes containing 1.0 mg of plasmid DNA, achieving levels of 7.4 ± 3.4 pM (range 0-16.4 pM), which was significantly greater than nontransfected (P = 0.045) and mock-transfected (P = 0.049) controls. Nevertheless, the level of hA₁AT measured in the ELF of transfected rats was well below the normal concentration in human lungs, and considerable variability in transgene expression was observed. The concentration of hA₁AT in ELF in healthy human subjects is approximately 0.9 ± 0.2 µM (17). Human A₁AT was also detected in the ELF of animals that received 0.2 and 2.0 mg of the complexed plasmid (2.4 ± 1.7 pM and 6.8 ± 5.3 pM of the human antiprotease, respectively). The hA₁AT in the BAL fluid must be from the PAM, because no detectable plasma levels were measured in any of the experimental animals (Figure 6). It is uncertain whether the secreted human protein was functional, because the assay for antiprotease activity was confounded by the presence of endogenous rat A₁AT in the ELF and was not quantitative for the human antiprotease.

View larger version (10K): [in this window] [in a new window]   Figure 6.   Detection of hA1AT in the pulmonary epithelial lining fluid of transfected rats. BAL samples were obtained from rats that received injections with transfection complexes containing increasing amounts of the plasmid pRcCMVhAAT (T 0.2 mg, T 1.0 mg, and T 2.0 mg); mannosylated polylysine bound to 1.0 mg of an irrelevant plasmid, pGL (ID); or polylysine bound to 1.0 mg of pRcCMVhAAT (IC) were assayed for hA1AT using ELISA, and compared with levels measured in nontransfected rats (NT ). No hA1AT was detected in the blood of any animal. The mean of hA1AT concentrations for each group is indicated by the solid line.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

Mannose-terminal glycoprotein conjugates have been shown to direct gene delivery to macrophages in vitro and in vivo by exploiting the mannose receptor. We report the successful transfer of the gene encoding hA₁AT into the lung via this receptor using a mannose-terminal conjugate, and the production of hA₁AT by the transfected cells. Specifically, macrophages in the alveolar and perivascular regions of the lungs of transfected rats expressed the hA₁AT transgene, and the human antiprotease was secreted into the ELF. Human A₁AT was not detected in the lungs of nontransfected and mock-transfected animals, although transcripts were found in lungs of three rats treated with the expression plasmid bound to poly-L-lysine by RT-PCR analysis. The alveoli are particularly important sites for correction because the deleterious effects of the NE are greatest in this region of the lung. Indeed, the decreased lung levels of the antiprotease in patients with A₁AT deficiency result in inadequate protection against lytic enzymes released by neutrophils because the alveolar walls are susceptible to even low levels of the elastase.

Because it is the primary site of production of A₁AT, the liver has been identified as a target for the transfer of the gene encoding hA₁AT (16, 23, 24). However, the alveocapillary membrane acts as a barrier to diffusion of A₁AT, causing a gradient in A₁AT levels between the blood and alveoli in humans (25). Pulmonary transfer of the hA₁AT gene should concentrate the antiprotease in the tissue most susceptible to injury caused by NE. Functional genes have been delivered to the lung using a variety of methods. Replication-deficient, recombinant adenoviruses successfully delivered the gene encoding hA₁AT to respiratory epithelial cells in cotton rats in vitro and in vivo (26). However, the level of hA₁AT detected in the ELF was determined to be well below the estimated threshold human protective level. Although the human antiprotease concentrations were not corrected for dilution, the absolute values of hA₁AT measured in the BAL collected from the transduced cotton rats were approximately 10-fold greater than the levels achieved in Sprague-Dawley rats using the mannose-terminal conjugate. The physiologic effects of treatment with adenovirus are unclear, though early generation adenoviral vectors administered at high viral titers produce a substantial inflammatory response in the lung (27). This adverse effect may be particularly undesirable in the treatment of A₁AT deficiency because proteases liberated by the recruited inflammatory cells could increase the amount of transgene product necessary to neutralize the protease.

Cationic liposomes have introduced expression plasmids to the lung. Brigham and colleagues reported that the chloramphenicol acetyltransferase reporter gene was successfully delivered to the lungs of mice by the intravenous infusion of liposome complexes (28). Zhu and associates found prolonged transgene expression in a variety of tissues in mice as long as 9 wk after a single intravenous injection of cationic liposome complexes (29). Macrophages and vascular endothelial cells were transfected, but parenchymal cells in the tissues also expressed the transgene. Canonico and coworkers have shown that the gene encoding hA₁AT could be delivered to the pulmonary vasculature and bronchial epithelium of rabbits by the intravenous infusion of cationic liposome complexes (30).

Moreover, the lungs have been targeted by the direct administration of liposome complexes. Certainly, inhaled liposome-DNA complexes could be specifically delivered to the conducting airways and alveoli. Cationic liposomes have been shown to mediate the delivery of exogenous, functional genes to the respiratory epithelium. The intratracheal instillation of cationic liposome-DNA complexes has resulted in transgene expression in the lungs of mice and rats. Stribling and coworkers bound plasmid DNA to a combination of neutral and cationic lipids and nebulized the resultant complexes into murine lungs (31). High levels of reporter-gene activity were detected in the conducting airway epithelia and alveolar pneumocytes. The hA₁AT gene complexed to cationic liposome (Lipofectin) was successfully introduced into airway epithelial cells and alveolar pneumocytes of rabbits using an aerosol delivery system, though the actual concentration of the human antiprotease was not measured in the ELF (30). Lipofection has been inefficient compared with the efficacy of gene delivery mediated by viral vectors. Although the newer lipid reagents may improve gene transfer, they also appear to have greater toxicity. The intratracheal administration of the newer liposome formulations to murine lungs have been shown to provoke a dose-dependent inflammatory reaction in the lungs (32). The proteases liberated by recruited inflammatory cells (i.e., neutrophils) could potentially increase the amount of transgene product necessary to neutralize the protease.

Receptor-mediated gene transfer provides specificity in the form of a noninfectious vector (33). The internalization of surface receptors is a common cellular response, and different receptors, such as the transferrin receptor (34) and polymeric immunoglobulin receptor (35), have been used to introduce exogenous genes into the lungs in vivo. Transferring the gene encoding hA₁AT into pulmonary macrophages may have several advantages. In contrast to many of the methods previously described which deliver the transgene to the more proximal airways, the mannose receptor permits the transfer of the hA₁AT gene to the alveoli, sites that are most vulnerable to destruction in patients with A₁AT deficiency. Proteins produced by transduced airway epithelial cells and secreted into the lumen poorly penetrate the epithelial barrier (36, 37). The epithelial barrier could prevent the transport of the antiprotease into the pulmonary interstitium, which is also potentially susceptible to the effects of neutrophil elastase. Transfected macrophages which reside in the interstitial space, however, may provide local protection against lytic enzymes. Finally, protease attack on the subcellular clefts of neutrophils and macrophages may affect the function of these inflammatory cells, and circulating A₁AT may not be accessible to these sites (10, 38). The local production of an exogenous A₁AT by cells deficient in the antiprotease could re-establish the protease-antiprotease balance in the pericellular environment and permit normal function of the inflammatory cell (10).

It is possible that the transfection complexes infused into the systemic circulation may be preferentially delivered to PAM, since the complexed DNA will "first pass" through the lungs. Nevertheless, although the mannose receptor is specific for tissue macrophages, it is not specific for the lung. Macrophages in the spleen also expressed the transgene, yet hA₁AT detected in the BAL fluid could not be due to diffusion from the blood because the human antiprotease was not found in the plasma. We suspect that extrapulmonary macrophages secrete hA₁AT, but the human antiprotease is undetectable because it is diluted by the blood volume. The inability of the molecular conjugate to selectively target the lungs may be critical for gene therapy because the indiscriminant transfer of the hA₁AT gene into other tissues may be contributing to the inefficiency of the method. The concentration of the hA₁AT measured in the ELF was subtherapeutic, well below the threshold level necessary to protect the lungs from the destructive effects of NE. Even within the lung, the sites of expression may not be optimal for delivery of hA₁AT. Transgene expression primarily occurred in the interstitial and perivascular macrophages of the lung, and not the alveolar space. PAM are functionally diverse, and the differences in gene transfer may be due to unequal expression of the mannose receptor on cells localized to the various regions of the lung.

Pulmonary alveolar macrophages bind mannose-terminal glycoproteins with high affinity and specificity. The ligands are delivered to pre-lysosomal acidic compartments and are ultimately trafficked to secondary lysosomes. Transgene expression, however, may be limited by the degradation of transfection complexes in lysosomes. It is possible that the efficiency of gene transfer through receptor-mediated endocytosis could be improved by interrupting the trafficking of transfection complexes. Erbacher and associates have shown that chloroquine, a lysosomotropic agent that interferes with the acidification of lysosomes and inhibits hydrolytic enzymes, enhances the expression of transgenes in human macrophages transfected with the glycosylated polylysine (12). Another pharmacologic agent, colchicine, which blocks the intracellular transport of endosomes along microtubules, improved the survival of DNA delivered via the asialoglycoprotein receptor pathway and increased transgene expression (39). Likewise, this drug could potentially augment gene transfer to macrophages via the mannose receptor. The addition of various endosomolytic agents to the molecular conjugate has also been shown to greatly enhance transgene expression. Replication-defective adenoviruses coupled to transferrin-based conjugates promote the release of transfection complexes from the endosomal compartment and prolong survival of the transgenes (40). Fusogenic peptides from other viruses, such as the influenza hemagglutinin HA-2, have a similar effect on transgene expression (41). However, the addition of such agents to molecular conjugates could have some disadvantages because disabled viruses or peptides may increase the immunogenicity of the complexes and limit their usefulness.

Because monosaccharides are poorer ligands for the mannose receptor than polyvalent glycoproteins, gene transfer efficiency into PAM may be enhanced by using an oligosaccharide, i.e., oligomannose, as a ligand (14, 42). An alternate monosaccharide substituted for mannose could also potentially increase the affinity of the DNA complexes because the mannose receptor recognizes other carbohydrate groups on glycoproteins, i.e., glucose, fucose, and N-acetylglucosamine (12, 43). In addition, a different receptor on macrophages could be targeted. The serpin enzyme complex (SEC) receptor has been used to successfully transfer genes to human hepatoma cells in culture (44). Abundant on the surface of hepatocytes and mononuclear phagocytes, the receptor recognizes a pentapeptide sequence that is exposed on serpins (serine protease inhibitors) after they bind their cognate proteases (45). The SEC receptor, however, may be ill-suited for the expression of an A₁AT transgene because it internalizes complexes of the antiprotease and elastase. It may be useful for the transfer of other genes to macrophages in the lung. For instance, directing gene transfer via the mannose receptor may be useful in delivering reagents to attack mycobacteria, which can defy destruction by residing dormant within macrophages for years (46, 47).

Thus mannose-terminal glycoprotein conjugates can direct functional genes to PAM by exploiting the mannose receptor. Although genes delivered via this receptor were expressed at low levels, this gene transfer technique appears to be a promising method for the introduction of transgenes to the lung.