Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization.

Vascular endothelial growth factor (VEGF) is important in pathological neovascularization, which is a key component of diseases such as the wet form of age-related macular degeneration, proliferative diabetic retinopathy and cancer. One of the most potent naturally occurring VEGF binders is VEGF receptor Flt-1. We have generated two novel chimeric VEGF-binding molecules, sFLT01 and sFLT02, which consist of the second immunoglobulin (IgG)-like domain of Flt-1 fused either to a human IgG1 Fc or solely to the CH3 domain of IgG1 Fc through a polyglycine linker 9Gly. In vitro analysis showed that these novel molecules are high-affinity VEGF binders. We have demonstrated that adeno-associated virus serotype 2 (AAV2)-mediated intravitreal gene delivery of sFLT01 efficiently inhibits angiogenesis in the mouse oxygen-induced retinopathy model. There were no histological observations of toxicity upon persistent ocular expression of sFLT01 for up to 12 months following intravitreal AAV2-based delivery in the rodent eye. Our data suggest that AAV2-mediated intravitreal gene delivery of our novel molecules may be a safe and effective treatment for retinal neovascularization.

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.

Adenoviral vector expressing ICP47 inhibits adenovirus-specific cytotoxic T lymphocytes in nonhuman primates.

Studies from several laboratories have shown that administration of E1-deleted Ad vectors results only in transient transgene expression in the lungs of immunocompetent animals. This is due, at least in part, to destruction of vector-transduced cells by host cellular immune responses (predominantly CD8(+) CTLs) directed against viral proteins and/or immunogenic transgene products. We have previously demonstrated that E1-deleted Ad vectors can lead to persistent expression of human cystic fibrosis transmembrane conductance regulator (hCFTR) in the lungs of several strains of immunocompetent mice, despite the presence of Ad-specific CTLs. However, we found that these same vectors gave rise only to transient hCFTR expression in the lungs of rhesus monkeys. We have constructed new Ad vectors that coexpress both hCFTR and the ICP47 gene from herpes simplex virus. ICP47 has been shown to inhibit the transporter associated with antigen presentation, thus blocking major histocompatibility antigen I (MHC class I)-mediated antigen presentation to CD8(+) T cells. The Ad/hCFTR/ICP47 vector decreased levels of cell-surface MHC class I molecules on infected monkey and human cell lines. Similar results were obtained with primary human cells and primary monkey airway epithelial cells. In vitro studies showed that the Ad/hCFTR/ICP47 vector decreased cytolysis by both monkey and human CTLs. When Ad/hCFTR/ICP47 was administered to the lungs of rhesus monkeys, it inhibited the generation of Ad-specific CTLs. However, natural killer cell activity was enhanced in monkeys treated with the Ad/hCFTR/ICP47 vector.

Scaleable chromatographic purification process for recombinant adeno-associated virus (rAAV).

BACKGROUND: Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Traditionally AAV has been purified from cell lysates using CsCl gradients; this approach however is not likely to be useful in large-scale manufacturing. Moreover gradient-purified AAV vectors tend to be contaminated with significant levels of cellular and adenoviral proteins and nucleic acid. To address the issue of purification we have developed a process scale method for the rapid and efficient purification of recombinant AAV (rAAV) from crude cellular lysates. METHODS: The preferred method for the purification of rAAVbetagal includes treatment of virally infected cell lysates with both trypsin and nuclease followed by ion exchange chromatography using ceramic hydroxyapatite and DEAE-Sepharose in combination with cellufine sulphate affinity chromatography. RESULTS: Purification of rAAV particles from crude cellular lysates co-infected with adenovirus was achieved using column chromatography exclusively. Column-purified rAAV was shown to be greater than 90% pure, free of any detectable contaminating adenovirus, biologically active, and capable of directing efficient gene transfer to the lungs of both cotton rats and mice. CONCLUSIONS: This study demonstrates the feasibility of using column chromatography alone for the isolation of highly purified rAAV vector. The methods described here are advancements in procedures to purify rAAV and are adaptable for commercial production of clinical-grade rAAV vector.

Modification of an adenoviral vector with biologically selected peptides: a novel strategy for gene delivery to cells of choice.

Recombinant adenoviruses are currently being used as vectors for gene delivery to a wide variety of cells and tissues. Although generally efficacious for gene transfer in vitro, improvement in the efficiency of vector delivery in vivo may aid several gene therapy applications. One major obstacle is the lack of high-affinity viral receptors on the surface of certain cells that are targets for gene therapy. In principle, incorporation of avid, cell-specific ligands into the virion could markedly improve vector entry into the desired tissues. We have developed a strategy for addressing this issue in the lung by biopanning differentiated, ciliated airway epithelial cells against a phage display library. The peptide with the most effective binding was coupled to the surface of an adenovirus using bifunctional polyethylene glycol (PEG) molecules. The chemically modified adenoviral vector was able to effect gene transfer to well-differentiated human airway epithelial cells by an alternative pathway dependent on the incorporated peptide. Coupling of PEG to the surface of the virus also served to partially protect the virus from neutralizing antibodies in vitro. These experiments will aid in the design of improved adenoviral vectors with the capacity for more specific and efficient delivery of therapeutic genes to desired target tissues. We have used a novel method for enhancing gene delivery to target cells by coupling a biologically selected peptide to the surface of an adenovirus with bifunctional PEG molecules. Modification of the viral capsid by the addition of a peptide with binding preference for differentiated ciliated airway epithelia allowed gene delivery to those cells by a novel entry pathway. Incorporation of the CFTR gene in a similarly modified vector resulted in correction of defective Cl- transport in well-differentiated epithelial cultures established from human cystic fibrosis (CF) donors. The presence of PEG molecules on the surface of the virus served, in addition, to reduce antibody neutralization. Modification of adenoviruses with PEG/peptide complexes can serve to partially overcome the barrier of inefficient gene transfer in some cell types and some of the adverse immunological responses associated with gene delivery by these vectors.

The extra- and intracellular barriers to lipid and adenovirus-mediated pulmonary gene transfer in native sheep airway epithelium.

Gene transfer to the respiratory epithelium is currently suboptimal and may be helped by the identification of limiting biological barriers. We have, therefore, developed an ex vivo model which retains many of the characteristics of in vivo native airways including mucociliary clearance, mucus coverage and an intact cellular structure. Using this model we have demonstrated several barriers to gene transfer. Liposome-mediated gene transfer was inhibited by normal mucus, with removal of this layer increasing expression approximately 25-fold. In addition both liposome and adenovirus were inhibited by CF sputum. The apical membrane represented a significant barrier to both agents. Adenovirus-mediated expression could be significantly augmented by increasing contact time or by pre-treatment of tissues with a nominally calcium-free medium. The presence of these extracellular and plasma membrane barriers appeared to be the key parameters responsible for the approximately three log difference in gene expression found in vitro compared with our ex vivo model. Cytoskeletal elements and the cell cycle also influenced in vitro gene transfer, and represent further barriers which need to be overcome.

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.

PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo.

Replication-defective recombinant adenovirus (Ad) vectors are under development for a wide variety of gene therapy indications. A potential limiting factor associated with virus gene therapy requiring repeated treatment is the development of a humoral immune response to the vector by the host. In animal models, there is a dose-dependent rise in neutralizing antibodies after primary vector administration, which can preclude effective repeat administration. The strategy we have developed to circumvent the neutralization of adenovirus vectors by antibodies is to mask their surface by covalent attachment of the polymer polyethylene glycol (PEG). Covalent attachment of PEG to the surface of the adenovirus was achieved primarily by using activated PEG tresylmonomethoxypolyethylene glycol (TMPEG), which reacts preferentially with the epsilon-amino terminal of lysine residues. We show that the components of the capsid that elicit a neutralizing immune response, i.e., hexon, fiber, and penton base, are also the main targets for PEGylation. Several protocols for PEGylation of an adenovirus vector were evaluated with respect to retention of virus infectivity and masking from antibody neutralization. We show that covalent attachment of polymer to the surface of the adenovirus can be achieved with retention of infectivity. We show further that PEG-modified adenovirus can be protected from antibody neutralization in the lungs of mice with high antibody titers to adenovirus, suggesting that PEGylation will improve the ability to administer Ad vectors on a repeated basis.

Novel role for E4 region genes in protection of adenovirus vectors from lysis by cytotoxic T lymphocytes.

Target cells infected with adenovirus (Ad) vectors containing intact E3 and E4 regions were found to be relatively resistant to lysis by Ad-specific cytotoxic T lymphocytes. Elements from both the E3 and the E4 regions were required for this effect, leading to the identification of a previously undescribed role for E4 gene products in resistance to cytolysis.

Adenovirus-mediated persistent cystic fibrosis transmembrane conductance regulator expression in mouse airway epithelium.

Replication-defective adenovirus (Ad) vectors have been used for gene transfer to the respiratory epithelium of experimental animals and individuals with cystic fibrosis. Studies from several laboratories have suggested that administration of first-generation Ad vectors results only in transient gene expression in the lung, due at least in part to destruction of vector-transduced cells by host cellular immune responses directed against viral proteins and/or immunogenic transgene products. We have constructed new Ad2-based, E1-deleted vectors encoding a weakly immunogenic transgene, the human cystic fibrosis transmembrane conductance regulator (hCFTR) under the control of the cytomegalovirus enhancer-promoter. These vectors contain wild-type E2 and E4 regions. These new Ad/CFTR vectors were instilled into the lungs of immunocompetent C57BL/6, BALB/c, and C3H mice. In vitro cytotoxic T lymphocyte (CTL) analysis indicated the presence of Ad-specific CTLs in treated mice. However, we were not able to demonstrate a CTL response specific for hCFTR. Reverse transcriptase PCR analysis demonstrated that hCFTR mRNA expression continued in all three strains of mice for at least 70 days, the last time point analyzed. The E3 region did not play a significant role in persistence of the Ad/CFTR vectors in the mouse lung. Functional hCFTR expression was also observed in the nasal epithelia of CF mutant mice. These results suggest that long-term expression of hCFTR is possible in the airway epithelia of immunocompetent mice without radical modification of Ad vector and in spite of the presence of CTLs.

Potentiation of gene transfer to the mouse lung by complexes of adenovirus vector and polycations improves therapeutic potential.

Recombinant adenovirus (Ad) vectors are being considered for in vivo delivery of various therapeutic genes. One limiting factor in the development of Ad-based gene therapy is the low efficiency of gene transfer to target tissues such as vascular endothelium, smooth muscle, and airway epithelium. Complexing Ad vector with various polycations has been shown to enhance transduction of cell lines otherwise resistant to Ad infection in vitro. On the basis of this observation, the activity of Ad/polycation complexes was tested in vivo in the mouse lung. The results indicated that several polycations were capable of enhancing transduction of mouse respiratory epithelium, leading to a 1-2 log increase in levels of transgene expression. Poly-L-lysine (PLL) and DEAE-dextran were examined further and were found to increase Ad-mediated gene transfer without any additional toxicity as assessed histologically or through the measurement of inflammatory cytokines in bronchoalveolar lavages. The two polycations also failed to affect the humoral response against Ad vector and were themselves nonimmunogenic under conditions leading to enhanced gene transfer. Moreover, the ability to use reduced doses of vector complexed with polycations resulted in lower levels of Ad-specific antibodies and, thereby, improved readministration of vector. These results suggest that complexing Ad vectors with polycations has the potential to improve the therapeutic index by increasing transgene expression while reducing unwanted responses associated with high doses of vector.

Adenovirus vector-infected cells can escape adenovirus antigen-specific cytotoxic T-lymphocyte killing in vivo.

The recent findings that prolonged expression of certain adenovirus (Ad) vector-encoded proteins, including human alpha1-antitrypsin (huAAT), mouse erythropoietin (EPO), and human factor IX, can be achieved in animals that do not mount an immune response to the reporter protein were obtained with mouse strains which have been shown to be capable of mounting a cellular immune response against Ad vector antigens. This suggests either that Ad vectors expressing nonimmunogenic transgenes fail to elicit a cellular immune response or that an Ad-specific cellular immune response does develop but is ineffective against cells expressing nonimmunogenic transgenes. Here we demonstrate that an Ad vector expressing huAAT administered by intravenous injection does stimulate an Ad-specific cellular immune response but that this response fails to abolish vector-directed gene expression in vivo. Moreover, expression of huAAT remained stable in animals stimulated by concurrent and multiple administrations of different Ad vectors or viruses. We also demonstrate prolonged expression of huAAT in CD1 mice transgenic for the huAAT gene, indicating that long-term expression is not restricted to C57BL/6 mice. These results demonstrate that under some circumstances, an Ad vector can direct prolonged expression of a nonimmunogenic transgene despite the presence of a robust Ad-specific cellular immune response.

Antibody to CD40 ligand inhibits both humoral and cellular immune responses to adenoviral vectors and facilitates repeated administration to mouse airway.

Adenoviral vectors have been used successfully to transfer the human CFTR cDNA to respiratory epithelium in animal models and to CF patients in vivo. However, studies done primarily in mice, indicate that present vector systems have limitations. Among other things, transgene expression in the lung is transient and the production of neutralizing antibodies against adenovirus correlates with a reduced ability to readminister a vector of the same serotype. Here we demonstrate that in mice, a transient blockade of costimulation between activated T cells and B cells/antigen presenting cells using a monoclonal antibody (MR1) against murine CD40 ligand inhibits the development of neutralizing antibodies to adenoviral (Ad) vector. MR1 also decreased the cellular immune response to Ad vector and allowed an increase in persistence of transgene expression. Furthermore, when administered with a second dose of Ad vector to mice preimmunized against vector, MR1 was able to interfere with the development of a secondary antibody response and allowed for high levels of transgene expression upon a third administration of vector to the airway.

Increased contact time improves adenovirus-mediated CFTR gene transfer to nasal epithelium of CF mice.

Multiple dosing with recombinant adenoviral vectors containing the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the nasal mucosa of cystic fibrosis (CF) transgenic mice reportedly results in only partial correction of the CF defect in chloride (Cl-) secretion without normalizing sodium (Na+) hyperabsorption, perhaps indicating inefficient gene transfer into the nasal airway epithelium in vivo. In this study, we have examined whether optimizing vector administration such as contact time could improve gene transfer efficiency. Changes in basal nasal potential difference (PD), and in PD (delta PD) following addition of amiloride and subsequent removal of Cl- from the luminal perfusate were assayed. As reported previously, the basal nasal PD was significantly more negative in CF mice (-24.9 +/- 2.1 mV) than in normal mice (-6.3 +/- 1.2 mV). Normal mouse nasal mucosa exhibited a large hyperpolarization in response to low Cl- substitution (delta PD of 8.5 +/- 1.9 mV), whereas the nasal mucosa of the CF mouse depolarized in response to this treatment. No correction of either the Cl- or Na+ transport defects were observed when 5 x 10(9) IU of Ad2/CFTR-5 were administered to the nasal passage of CF mice over a period of 5-20 min. However, when CF mice were perfused over a period of 60 min with the same dose of vector, a significant response (delta PD of 5.9 +/- 1.1 mV) to low Cl- substitution was detected 2 days later. In these mice, the basal nasal PD (-10.5 +/- 1.4 mV) and the response to amiloride were also reduced, indicating a partial correction of the Na+ transport defect. Expression of functional CFTR activity was transient with no measurable delta PD signals observed by day 7 post-treatment. These results suggest that prolonging the contact between an adenoviral vector and the respiratory epithelium enhances the efficiency of gene transfer and can result in improved correction of the CF Na+ and Cl- ion transport defects. Therefore, strategies that improve internalization of viral vectors and that prolong their contact time with target cells may result in the improved clinical efficacy of such vectors.

Analysis of recombinant adeno-associated virus packaging and requirements for rep and cap gene products.

Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Because the gene transfer vector commonly retains only the AAV terminal repeats, propagation of recombinant AAV (rAAV) requires that the viral replication (Rep) and capsid (Cap) proteins be supplied in trans. In an effort to optimize the production of these vectors, a panel of helper plasmids was constructed to determine if expression of the rep and/or cap genes is a limiting factor for rAAV packaging. Expression of the Rep and Cap proteins was increased by replacing the endogenous AAV promoters, p5 and p40, with the Rous sarcoma virus (RSV) long terminal repeat (LTR) and the cytomegalovirus immediate-early promoter, respectively. Increased synthesis of the Cap proteins resulted in an approximately 10-fold increase in the yield of rAAV, indicating that production of capsid proteins is one limiting factor for rAAV packaging. Expression of the rep gene from the RSV LTR not only failed to increase the yield of rAAV but also prevented activation of p40 transcription with adenovirus infection, resulting in a reduced level of capsid protein synthesis.

Generation of cytotoxic T lymphocytes against immunorecessive epitopes after multiple immunizations with adenovirus vectors is dependent on haplotype.

Currently, adenovirus (Ad) is being considered as a vector for the treatment of cystic fibrosis as well as other diseases. However, the cytotoxic T lymphocyte (CTL) response to Ad could limit the effectiveness of such approaches. Since the CTL response to virus infection is often focused on one or a few immunodominant epitopes, one approach to circumvent this response is to create vectors that lack these immunodominant epitopes. The effectiveness of this approach was tested by immunizing mice with human group C adenoviruses. Three mouse strains (C57BL/10SnJ [H-2b], C3HeB/FeJ [H-2k], and BALB/cByJ [H-2d]) were immunized with wild-type Ad or Ad vectors lacking the immunodominant antigen(s), and the CTL responses were measured. In C57BL/10 (B10) mice, a single inoculation intraperitoneally (i.p.) led to the recognition of an immunodominant antigen in E1A. When B10 mice were inoculated multiple times either i.p. or intranasally with wild-type Ad or an Ad vector lacking most of the E1 region, subdominant epitopes outside this region were recognized. In contrast, C3H mice inoculated with wild-type Ad recognized an epitope mapping within E1B. When inoculated twice with Ad vectors lacking both E1A and E1B, no immunorecessive epitopes were recognized. The immune response to Ad in BALB/c mice was more complex. CTLs from BALB/c mice inoculated i.p. with wild-type Ad recognized E1B in the context of the major histocompatibility complex (MHC) class I Dd allele and a region outside E1 associated with the Kd allele. When BALB/c mice were inoculated with E1-deleted Ad vectors, only the immunodominant Kd-restricted epitope was recognized, and Dd-restricted CTLs did not develop. This report indicates that the emergence of CTLs against immunorecessive epitopes following multiple administrations of Ad vectors lacking immunodominant antigens is dependent on haplotype and could present an obstacle to gene therapy in an MHC-diverse human population.

Effect of the E4 region on the persistence of transgene expression from adenovirus vectors.

The utility of adenovirus vectors for gene therapy is limited by the transience of expression that has been observed in various in vivo models. Immunological responses to viral targets can eliminate transduced cells and cause the loss of transgene expression. We previously described the characterization of an E4 modified adenovirus, Ad2E4ORF6, which is replication defective in cotton rats. We reasoned that gene transfer vectors based on Ad2E4ORF6 would have a reduced potential for viral gene expression in vivo which might be beneficial for achieving persistence of transgene expression. E1 replacement vectors expressing the cystic fibrosis transmembrane regulator or beta-galactosidase were constructed as series of vectors that differed with respect to the E4 region. Vectors containing a wild-type E4 region, E4 open reading frame 6, or a complete E4 deletion were compared in the lungs of BALB/c mice for persistence of expression. Results obtained with nude mice indicate that nonimmunological factors have a major influence on the longevity of transgene expression. Expression was transient from the E1a promoter with all vectors but persisted from the cytomegalovirus promoter only with a vector containing a wild-type E4 region. Transience of expression did not correlate with the disappearance of vector DNA, suggesting that promoter down-regulation may be involved. Coinfection studies indicate an E4 product(s) could be supplied in trans to allow persistent expression from the cytomegalovirus promoter. In summary, the choice of promoter is important for achieving persistence of expression; in addition, some promoters are highly influenced by the context of the vector backbone.

Safety of airway gene transfer with Ad2/CFTR2: aerosol administration in the nonhuman primate.

In this study, the safety and efficacy of aerosol delivery to non-human primates of an adenoviral vector encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) were evaluated. The technique of concurrent flow spirometry was used to determine the deposited dose of Ad2/CFTR-2, which ranged from 3 to 8 x 10(10) I.U. Transgene DNA was detected by the polymerase chain reaction (PCR) in lung tissue from all treated animals, and human CFTR mRNA was detected on days 3, 7, and 21 post-exposure. The treatment was well tolerated, with no evidence of respiratory distress. Histologic changes in the lungs from Ad2/CFTR-2-treated animals were mild and, overall, indistinguishable from animals exposed to aerosolized vehicle. One vector-treated animal demonstrated an increase in lavage lymphocyte numbers 3 days after treatment and another had an abnormal chest radiograph 14 days after treatment. A third vector-treated animal had histologic evidence of a bronchointerstitial pneumonia 7 days after aerosol treatment that resolved by day 21. This study demonstrated that Ad2/CFTR-2 can effectively be delivered to the lungs of nonhuman primates and result in minimal adverse effects.