Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. II. Transfection efficiency in airway epithelium.

A phase I clinical trial was conducted in which recombinant adenovirus containing the cystic fibrosis trans-membrane regulator (CFTR) (Ad2/CFTR) was administered by bronchoscopic instillation or aerosolization to the lungs of cystic fibrosis (CF) patients. In this paper, we evaluate the efficiency of Ad2/CFTR-mediated transduction of bronchial airway cells. The ability of an Ad2/CFTR vector to transduce airway cells was first evaluated in patients to whom the vector was administered by bronchoscopic instillation. Cells at the administration site were collected 2 days after treatment by bronchoscopic brushing. Ad2-specific CFTR DNA was detected in four of five individuals by PCR, and Ad2-specific CFTR RNA was detected in three of five individuals by RT-PCR. Ad2/CFTR-mediated transduction of airway epithelial cells was then determined in CF individuals receiving this vector by aerosol inhalation. Ad2-specific CFTR DNA was detected in 13 of 13 individuals 2 days after aerosolization, and in 3 of 5 individuals 7 days after aerosolization. Ad2-specific RNA was detected in 4 of 13 individuals on day 2, but was not detected in the 5 individuals tested on day 7. The percentage of airway epithelial cells containing nuclear-localized vector DNA was < or =2.4% as determined by fluorescence in situ hybridization (FISH). However, in some cases, a high percentage of nonepithelial mononuclear cells or squamous metaplastic epithelial cells was infected with the adenoviral vector. In conclusion, aerosol administration is a feasible means to distribute adenoviral vectors throughout the conducting airways, but improvements in adenovirus-mediated transduction of airway epithelial cells are necessary before gene therapy for CF will be effective.

Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. I. Methods, safety, and clinical implications.

Cystic fibrosis (CF), an autosomal recessive disorder resulting from mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene, is the most common lethal genetic illness in the Caucasian population. Gene transfer to airway epithelium, using adenoviruses containing normal CFTR cDNA, leads to transient production of CFTR mRNA and, in some studies, to correction of the airway epithelial ion transport defect caused by dysfunctional CFTR. Inflammatory responses to the adenoviral vector have been reported, particularly at high viral titers. We evaluated the effects of adenovirus-mediated CFTR gene transfer to airway epithelium in 36 subjects with CF (34 individuals, 2 of whom received two separate doses of vector), 20 by lobar instillation and 16 by aerosol administration. Doses ranged from 8 x 10(6) to 2.5 x 10(10) infective units (IU), in 0.5-log increments. After lobar administration of low doses there were occasional reports of cough, low-grade temperature, and myalgias. At the highest lobar dose (2.5 x 10(9) IU) two of three patients had transient myalgias, fever, and increased sputum production with obvious infiltrates on CT scan. After aerosol administration there were no significant systemic symptoms until the 2.5 x 10(10) IU dose, when both patients experienced myalgias and fever that resolved within 24 hr. There were no infiltrates seen on chest CT scans in any of the patients in the aerosol administration group. There were no consistent changes in pulmonary function tests or any significant rise in serum IgG or neutralizing antibodies in patients from either group. Serum, sputum, and nasal cytokines, measured before and after vector administration, showed no correlation with adenoviral dose. Gene transfer to lung cells was inefficient and expression was transient. Cells infected with the vector included mononuclear inflammatory cells as well as cuboidal and columnar epithelial cells. In summary, we found no consistent immune response, no evidence of viral shedding, and no consistent change in pulmonary function in response to adenovirus-mediated CFTR gene transfer. At higher doses there was a mild, nonspecific inflammatory response, as evidenced by fevers and myalgias. Overall, vector administration was tolerated but transfer of CFTR cDNA was inefficient and transgene expression was transient for the doses and method of administration used here.

A clinical inflammatory syndrome attributable to aerosolized lipid-DNA administration in cystic fibrosis.

Immunologic reactivity to lipid-DNA conjugates has traditionally been viewed as less of an issue than with viral vectors. We performed a dose escalation safety trial of aerosolized cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the lower airways of eight adult cystic fibrosis patients, and monitored expression by RT-PCR. The cDNA was complexed to a cationic lipid amphiphile (GL-67) consisting of a cholesterol anchor linked to a spermine head group. CFTR transgene was detected in three patients at 2-7 days after gene administration. Four of the eight patients developed a pronounced clinical syndrome of fever (maximum of 103.3EF), myalgias, and arthralgia beginning within 6 hr of gene administration. Serum IL-6 but not levels of IL-8, IL-1, TNF-alpha, or IFN-gamma became elevated within 1-3 hr of gene administration. No antibodies to the cationic liposome or plasmid DNA were detected. We found that plasmid DNA by itself elicited minimal proliferation of peripheral blood mononuclear cells taken from study patients, but led to brisk immune cell proliferation when complexed to a cationic lipid. Lipid and DNA were synergistic in causing this response. Cellular proliferation was also seen with eukaryotic DNA, suggesting that at least part of the immunologic response to lipid-DNA conjugates is independent of unmethylated (E. coli-derived) CpG sequences that have previously been associated with innate inflammatory changes in the lung.

Inhibitory effect of cystic fibrosis sputum on adenovirus-mediated gene transfer in cultured epithelial cells.

Effective gene transfer to the airway epithelial cells of individuals with cystic fibrosis (CF) requires gene therapy vectors to effectively penetrate the mucous lining of the airways of these patients. In this study, we examined the effects of the aqueous sol fraction of sputum recovered from CF patients (CF sol) on adenovirus (Ad)-mediated gene transfer to cultured epithelial cells. Sputum collected from patients with CF was separated into aqueous sol and gel fractions by ultracentrifugation and the sol fraction from different individuals was pooled. To determine if CF sol affects Ad-mediated transfection, Fisher rat thyroid (FRT) epithelial cells or normal human bronchial epithelial (NHBE) cells were infected with an Ad encoding beta-galactosidase (Ad2/betagal-2) in the presence or absence of the pooled CF sol. Transfection efficiency was determined by measuring beta-Gal activity. CF sol significantly inhibited Ad2-mediated gene transfer in a dose-dependent manner when the vector was incubated with CF sol prior to exposure to the cells. In contrast, preincubation of the cells with the sol was without effect. The inhibition of Ad-mediated gene transfer by CF sol was not related to its low pH, was abrogated by preadsorption with an Ad2 serotype vector, and was neutralized by heat treatment, but was not affected by treatment with protease inhibitors. Analysis of CF sol fractions from seven different individuals with CF showed inhibition of Ad-mediated gene transfer in four of the seven samples tested and, further, the inhibitory effect was correlated with the presence of Ad-specific antibodies. We conclude that preexisting adenovirus-specific antibodies present in some of the patient samples were the predominant factor inhibiting Ad-mediated gene transfer.

Immunogene therapy for murine melanoma using recombinant adenoviral vectors expressing melanoma-associated antigens.

Adenoviral vectors expressing tumor-associated antigens can be used to evoke a specific immune response and inhibit tumor growth. In this study, we tested the efficacy of adenoviral vectors encoding human gp100 (Ad2/hugp100), murine gp100 (Ad2/mugp100), or murine TRP-2 (Ad2/muTRP-2) for their ability to elicit a specific cellular immune response and inhibit the growth of B16 melanoma tumor cells in the mouse. C57BL/6 mice were immunized with Ad2/hugp100, Ad2/mugp100, or Ad2/muTRP-2 either 2 weeks prior to B16-F10 tumor challenge (prophylactic treatment) or 3 days after tumor challenge (active treatment). Ad2/hugp100 and Ad2/muTRP-2 administered to two or more intradermal (i.d.) sites inhibited subsequent subcutaneous tumor growth in > or = 80% of the mice and elicited an antigen-specific cytotoxic T lymphocyte response, whereas other administration routes were not as effective. Ad2/mugp100 administered to two i.d. sites did not inhibit tumor growth or provoke cellular immunity. Immunization was less effective with active treatment where tumor growth was not significantly inhibited by a single dose of either Ad2/muTRP-2 or Ad2/hugp100. However, increasing the number of intradermal immunization sites and the number of doses resulted in progressive improvements in protection from tumor growth in the active treatment model. In conclusion, breaking host tolerance to elicit protective immunity by using adenoviral vectors expressing melanoma-associated antigens is dependent upon the choice of antigen, the site of administration, and the number of doses. These observations provide insights into the clinical applicability of adenoviral vaccines for immunotherapy of malignant diseases.

E4ORF3 requirement for achieving long-term transgene expression from the cytomegalovirus promoter in adenovirus vectors.

Analysis of transgene expression under the control of the cytomegalovirus (CMV) promoter from adenovirus vectors in which the E4 region was modified indicated that E4ORF3 is required for long-term expression in the murine lung. CMV promoter truncation led to the persistence of expression in the absence of E4, thus eliminating the ORF3 requirement.

Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo.

Cationic lipids show promise as vectors for transfer of CFTR cDNA to airway epithelia of patients with cystic fibrosis (CF). However, previous studies have not compared the effect of DNA-lipid to DNA alone. Recently, we developed a formulation of plasmid encoding CFTR (pCF1-CFTR) and cationic lipid (GL-67:DOPE) that generated greater gene transfer in mouse lung than previously described DNA-lipid vectors. Therefore, we tested the hypothesis that DNA-lipid complexes were more effective than DNA alone at transferring CFTR cDNA to airway epithelia in vivo. We administered complexes of DNA-lipid to one nostril and DNA alone to the other nostril in a randomized, double-blind study. Electrophysiologic measurements showed that DNA-lipid complexes partially corrected the Cl- transport defect. Importantly, the pCF1-CFTR plasmid alone was at least as effective as complexes of DNA with lipid. Measurements of vector-specific CFTR transcripts also showed gene transfer with both DNA-lipid and DNA alone. These results indicate that nonviral vectors can transfer CFTR cDNA to airway epithelia and at least partially restore the Cl- transport defect characteristic of CF. However, improvements in the overall efficacy of gene transfer are required to develop a treatment for CF.

Quantitation of fibroblast population growth rate in situ using computerized image analysis.

The development of computer-assisted image analysis has provided the technology to rapidly determine the population size of cultured cell monolayers in situ. We have adapted this technology to determine the population growth rate of cultured fibroblasts for use in a high-replicate format. Human lung fibroblasts were seeded into 1/2 A 96-well plates that had one-half the culture area of standard 96-well plates. The cells were cultured in medium supplemented with different concentrations of FBS and on days 0, 1, 2, 3, 5, and 7, and their nuclei were stained with propidium iodide. A microscopic field representing one-quarter of a well of fluorescent nuclear images was captured onto a Macintosh computer, and the number of nuclei were counted using an image analysis software program. There were no significant differences between the number of nuclei counted manually and the number counted using computer-assisted software, until day 7 where the cells were multilayered (P < 0.05). This image analysis method was compared to other assays typically used to estimate cell proliferation or population size, namely hemocytometer counting, a rapid colorimetric staining assay using naphthol blue-black, and [3H]-thymidine incorporation. The growth rates derived using image analysis were in close agreement with results derived from hemocytometer counts and [3H]-thymidine incorporation. However, the growth rates of cells grown in high concentrations of FBS as determined using naphthol blue-black were substantially lower than results from image analysis. We conclude that this adaptation of computer-assisted image analysis provides a method to derive accurate growth curves by directly counting the number of cells in a large number of replicates.

Effects of dibutyrylcyclic adenosine monophosphate on bleomycin-induced lung toxicity in hamsters.

Cyclic nucleotides play an important role in the regulation of fibroblast proliferation and collagen metabolism. In the present study, the antifibrotic potential of dibutyrylcAMP (Bt2cAMP) was evaluated in the bleomycin (BLM)-hamster model of pulmonary fibrosis. Bt2cAMP (10 mg kg-1, s.c.) or saline (SA, s.c.) was given daily two days prior to the first intratracheal (i.t.) dose of BLM or SA and thereafter throughout the study. BLM or SA was instilled i.t. in three consecutive doses (2.5, 2.0 and 1.5 U 5ml-1 kg-1) at weekly intervals. Hamsters were killed at 7, 14 and 20 days after the third i.t. instillation. Bt2cAMP significantly reduced the contents of lung hydroxyproline and lung thiobarbituric acid reactive substance equivalents in BLM-treated animals at 7 and 14 days. Bt2cAMP significantly elevated lung superoxide dismutase activity in BLM-treated animals at 7 days. Lung prolyl hydroxylase activity was significantly elevated at 14 and 20 days in SABLM- and Bt2cAMPBLM-treated animals. The ratio of cAMP/cGMP was significantly reduced at all time points in SABLM-treated animals but only at 7 and 14 days in Bt2cAMPBLM-treated animals. Bt2cAMP caused no significant changes in lung calcium and calmodulin levels and protein content of the bronchoalveolar lavage. BLM significantly increased various inflammatory cell counts in the lavage at all three time points. The cell counts in the Bt2cAMPBLM groups were generally lower at 7 days and higher at 20 days than those of the SABLM groups. Histological evaluation showed that the lungs of Bt2cAMPBLM-treated hamsters progressed from an inflammatory cell lesion to a fibrotic lesion at a slower rate than the SABLM groups. It was concluded that Bt2cAMP attenuated BLM-induced pulmonary fibrosis in hamsters in part by delaying the acute phase of the inflammatory reaction.

Double-stranded RNA regulation of DNA synthesis in fibroblasts.

The double-stranded RNA molecule polyinosinic-polycytidylic acid (poly IC) has been found in some studies to have a mitogenic effect on fibroblast proliferation while other studies found poly IC to have an inhibitory effect on proliferation. In this study, we investigated whether a stabilized form of poly IC complexed with poly-L-lysine and carboxymethylcellulose (poly ICLC) had a bidirectional effect on DNA synthesis in fibroblasts from four different cell lines and determined factors that potentially influence this bidirectional effect. In medium containing fetal bovine serum, poly ICLC slightly increased the levels of [3H]thymidine incorporation in growing fibroblasts in three of the four fibroblast cell lines tested, while poly ICLC increased [3H]thymidine incorporation in confluent, quiescent fibroblasts in two of four cell lines. Poly ICLC did not induce DNA synthesis in subconfluent, quiescent or in confluent, quiescent fibroblasts under serum-free conditions. Poly ICLC significantly suppressed serum-induced [3H]thymidine incorporation by quiescent fibroblasts in all cell lines. We conclude that the stimulatory and inhibitory effects of poly ICLC on DNA synthesis are influenced by both the cell line and the presence of serum components in the culture medium but not by population density.

Ultrastructure of pericytes in early stages of histamine-induced inflammation.

Physiological and ultrastructural assessment of changes in the walls of venules in the rat cremaster muscle after administration of histamine indicates that pericytes have essential roles in the normal functioning of venules during inflammation. Fluorescein-labelled albumin was used to quantitate macromolecular leakage and to select suitable venules for ultrastructural analysis 4 and 7 minutes after addition of histamine. Pericytes were concentrated over endothelial cell junctions and gaps. At 4 minutes, when albumin leakage was becoming detectable, gaps between endothelial cells were observed in the venule wall. In 24 serially sectioned gaps, pericytes formed covers, with contact points to the endothelial cells along the sides of the gaps. At 7 minutes, when albumin leakage was maximal, gaps with pericyte covers were still evident, but more commonly observed were pericyte covers over closed endothelial cell junctions. Spaces between the innermost pericytes and endothelial cells were enlarged by an order of magnitude, from 95 nm in controls to 872 nm at 4 minutes and 958 nm at 7 minutes. Pericytes formed coverings or bridges over inclusions of extravasated cells, fluid, proteins, and the vascular label monastral blue. The data indicate that pericytes protect the endothelial lining of venules during histamine-induced inflammation by forming a cohesive covering across gaps.