Novel GP64 envelope variants for improved delivery to human airway epithelial cells.

Lentiviral vectors pseudotyped with the baculovirus envelope protein GP64 transduce primary cultures of human airway epithelia (HAE) at their apical surface. Our goal in this study was to harness a directed evolution approach to develop a novel envelope glycoprotein with increased transduction properties for HAE. Using error-prone PCR, a library of GP64 mutants was generated and used to prepare a diverse pool of lentiviral virions pseudotyped with GP64 variants. The library was serially passaged on HAE and three GP64 mutations were recovered. Single-, double- and the triple-combination mutant envelope glycoproteins were compared with wild-type GP64 for their ability to transduce HAE. Our results suggest that lentiviral vectors pseudotyped with evolved GP64 transduced HAE with greater efficiency than wild-type GP64. This effect was not observed in primary cultures of porcine airway epithelial cells, suggesting that the directed evolution protocol was species specific. In summary, our studies indicate that serial passage of a GP64 mutant library yielded specific variants with improved HAE cell tropism, yielding tools with the potential to improve the success of gene therapy for airway diseases.

A novel gene delivery method transduces porcine pancreatic duct epithelial cells.

Gene therapy offers the possibility to treat pancreatic disease in cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene; however, gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF; thus, CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene-transfer vectors. Our objective is to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. We catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green-fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous (IV) injection of AAV9CMV.sceGFP did not transduce the pancreas. Our technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via the umbilical artery in newborns and via the femoral artery at older ages--delivery approaches that can be translated to humans.

Progress and prospects: prospects of repeated pulmonary administration of viral vectors.

Pulmonary gene therapy may ultimately cure diseases such as cystic fibrosis, alpha1-antitrypsin deficiency, lung cancer and pulmonary hypertension. Efficient expression of delivered genes in target cell types is essential for the achievement of this goal. To this end, re-administration of viral vectors may be required (1) to increase the percentage of transduced airway epithelial cells, (2) to direct gene transfer to individual lobes during successive delivery sessions or (3) to boost attenuated expression over time. Immune responses to viral proteins or viral-encoded proteins are the greatest barrier to repeated vector administration.

Monocytic/macrophagic pneumonitis after intrabronchial deposition of vascular endothelial growth factor in neonatal lambs.

Preterm and young neonates are prone to inadequate surfactant production and are susceptible to respiratory distress syndrome characterized by alveolar damage and hyaline-membrane formation. Glucocorticoid therapy is commonly used in preterm and young infants to enhance lung maturation and surfactant synthesis. Recently, vascular endothelial growth factor (VEGF) was suggested to be a novel therapeutic agent for lung maturation that lacked adverse effects in mice. The purpose of this study was to assess the safety of incremental concentration (0.0005, 0.005, and 0.05 mg/ml) and duration (16, 24, and 32 hours) of recombinant human VEGF after bronchoscopic instillation (10 ml) in neonatal lambs. High-dose VEGF caused locally extensive plum-red consolidation that was microscopically characterized by interstitial and alveolar infiltrates of cells that were morphologically and phenotypically (CD68+) consistent with monocytes/macrophages. T cells (CD3+) and B cells (CD79+) were located primarily in bronchus/bronchiole-associated lymphoid tissue and were not consistently altered by treatment with VEGF. The dose of VEGF had significant effects on both gross lesions (P < .0047) and microscopic monocyte/macrophage recruitment scores (P < .0001). Thus, the VEGF dose instilled into the lung greatly influenced cellular recruitment and lesion development. The post-dosing interval of VEGF in this study had minor impact (no statistical significance) on cellular recruitment. This study showed that airway deposition of VEGF in the neonatal lamb induces monocyte/macrophage recruitment to the lung and high doses can cause severe lesions. The cellular recruitment suggests further research is needed to define dosages that are efficacious in enhancing lung maturation while minimizing potential adverse effects.

Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors--design, biosafety, and production.

Replication defective vectors derived from simple retroviruses or the more complex genomes of lentiviruses continue to offer the advantages of long-term expression, cell and tissue specific tropism, and large packaging capacity for the delivery of therapeutic genes. The occurrence of adverse events caused by insertional mutagenesis in three patients in a gene therapy trial for X-linked SCID emphasizes the potential for problems in translating this approach to the clinic. Several genome-wide studies of retroviral integration are now providing novel insights into the integration site preferences of different vector classes. We review recent developments in vector design, integration, biosafety, and production.

Expression and regulation of novel human beta-defensins in gingival keratinocytes.

This study evaluated the expression and regulation of beta-defensins DEFB-104 and the recently identified DEFB-105-14 in gingival keratinocytes. Keratinocytes from healthy subjects were exposed to cytokines, Escherichia coli lipopolysaccharide or Candida species. Total RNA was extracted and defensin expression analyzed by reverse transcription-polymerase chain reaction. Three patterns of expression were seen: no expression, constitutive expression and inducible expression. Constitutive mRNA expression was evident for DEFB-104, 107, 109, 111, and 112. DEFB-108 and 114 were induced by interleukin (IL)-1beta and Candida species. For DEFB-108 expression, synergism was observed when IL-1beta was combined with tumor necrosis factor-alpha or interferon-gamma. Downregulation of DEFB-109 occurred following treatment with Candida albicans. These findings suggest a role for multiple beta-defensins in response to oral infection. Further investigation is needed to better understand their function, both in terms of antimicrobial activities and contributions to innate and acquired immunity.

Apical barriers to airway epithelial cell gene transfer with amphotropic retroviral vectors.

Gene transfer to airway epithelia with amphotropic pseudotyped retroviral vectors is inefficient following apical vector application. To better understand this inefficiency, we localized the expression of Pit2, the amphotropic receptor, in polarized human airway epithelia. Pit2 was expressed on both the apical and basolateral surfaces of the cells, suggesting that factors other than receptor abundance may limit apical gene transfer efficiency. Binding studies performed with radiolabeled amphotropic MuLV suggested that the apically applied virus binds to Pit2. Hypothetical barriers to retroviral gene transfer include the apical glycocalyx and other secreted products of epithelia. In this study, we demonstrated that sialic acid, keratan sulfate and collagen type V are present on the apical surface of well-differentiated human airway epithelia. While enzyme treatment reduced the abundance of these components, the treatment also decreased the transepithelial resistance to approximately 35% of the controls, suggesting that the epithelial integrity was impaired. To attain an airway epithelial culture with a modified apical surface and intact epithelial integrity, we utilized 100 mM 2-deoxy-D-glucose, a glycosylation inhibitor, to prevent the glycocalyx from reforming following enzyme treatment. This approach allowed the resistance, but not the apical glycocalyx to recover. Despite this physical modification of the cell surface, the amphotropic retroviral vector failed to transduce airway epithelia following apical application. These results suggest that factors other than apical receptor abundance and the glycocalyx inhibit amphotropic retroviral gene transfer in human airway epithelia.

Cathelicidin peptides inhibit multiply antibiotic-resistant pathogens from patients with cystic fibrosis.

Endogenous peptide antibiotics are under investigation as inhaled therapeutic agents for cystic fibrosis (CF) lung disease. The bactericidal activities of five cathelicidin peptides (LL37 [human], CAP18 [rabbit], mCRAMP [mouse], rCRAMP [rat], and SMAP29 [sheep]), three novel alpha-helical peptides derived from SMAP29 and termed ovispirins (OV-1, OV-2, and OV-3), and two derivatives of CAP18 were tested by broth microdilution assays. Their MICs were determined for multiply antibiotic-resistant Pseudomonas aeruginosa (n = 24), Burkholderia cepacia (n = 5), Achromobacter xylosoxidans (n = 5), and Stenotrophomonas maltophilia (n = 5) strains isolated from CF patients. SMAP29 was most active and inhibited mucoid and nonmucoid P. aeruginosa strains (MIC, 0.06 to 8 microg/ml). OV-1, OV-2, and OV-3 were nearly as active (MIC, 0.03 to 16 microg/ml), but CAP18 (MIC, 1.0 to 32 microg/ml), CAP18-18 (MIC, 1.0 to >32 microg/ml), and CAP18-22 (MIC, 0.5 to 32 microg/ml) had variable activities. LL37, mCRAMP, and rCRAMP were least active against the clinical isolates studied (MIC, 1.0 to >32 microg/ml). Peptides had modest activities against S. maltophilia and A. xylosoxidans (MIC range, 1.0 to > 32 microg/ml), but none inhibited B. cepacia. However, CF sputum inhibited the activity of SMAP29 substantially. The effects of peptides on bacterial cell membranes and eukaryotic cells were examined by scanning electron microscopy and by measuring transepithelial cell resistance, respectively. SMAP29 caused the appearance of bacterial membrane blebs within 1 min, killed P. aeruginosa within 1 h, and caused a dose-dependent, reversible decrease in transepithelial resistance within 5 h. The tested cathelicidin-derived peptides represent a novel class of antimicrobial agents and warrant further development as prophylactic or therapeutic agents for CF lung disease.

Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappa B.

Reactive oxygen species (ROS) are important second messengers generated in response to many types of environmental stress. In this setting, changes in intracellular ROS can activate signal transduction pathways that influence how cells react to their environment. In sepsis, a dynamic proinflammatory cellular response to bacterial toxins (e.g. lipopolysaccharide or LPS) leads to widespread organ damage and death. The present study demonstrates for the first time that the activation of Rac1 (a GTP-binding protein), and the subsequent production of ROS, constitutes a major pathway involved in NFkappaB-mediated tumor necrosis factor-alpha (TNFalpha) secretion following LPS challenge in macrophages. Expression of a dominant negative mutant of Rac1 (N17Rac1) reduced Rac1 activation, ROS formation, NFkappaB activation, and TNFalpha secretion following LPS stimulation. In contrast, expression of a dominant active form of Rac1 (V12Rac1) mimicked these effects in the absence of LPS stimulation. IKKalpha and IKKbeta were both required downstream modulators of LPS-activated Rac1, since the expression of either of the IKK dominant mutants (IKKalphaKM or IKKbetaKA) drastically reduced NFkappaB-dependent TNFalpha secretion. Moreover, studies using CD14 blocking antibodies suggest that Rac1 induces TNFalpha secretion through a pathway independent of CD14. However, a maximum therapeutic inhibition of LPS-induced TNFalpha secretion occurred when both CD14 and Rac1 pathways were inhibited. Our results suggest that targeting both Rac1- and CD14-dependent pathways could be a useful therapeutic strategy for attenuating the proinflammatory cytokine response during the course of sepsis.

Ontogeny of CLCN3 chloride channel gene expression in human pulmonary epithelium.

Human fetal bronchopulmonary epithelia secrete liquid, and this chloride (Cl)-dependent process is important for normal lung growth. At the time of birth there is a maturational transition from a secretory to an absorptive phenotype. The pathways for Cl exit from the apical membrane which are required for fetal lung liquid secretion are unknown but are thought to be independent of the cystic fibrosis transmembrane conductance regulator. We determined the ontogeny of expression of the CLCN family of voltage-dependent Cl channel genes (CLCN2 through 6, K(a) and K(b)) in the human lung to identify potential pathways for pulmonary liquid secretion. Only CLCN3 and CLCN6 messenger RNA were detected by Northern analysis of fetal whole lung tissue. Ribonuclease protection assays confirmed the expression of CLCN3 and also revealed expression of CLCN2. The ontogeny of expression of these two channels was similar, peaking in midgestation and declining postnatally. In situ hybridization localized the CLCN2 and CLCN3 messages to airway and distal pulmonary epithelia and to pulmonary blood vessels. We conclude that CLCN3 is expressed in human airway epithelia and expression is developmentally regulated. The contribution of these channels to pulmonary epithelial liquid transport and lung development remains to be determined.

Infection of human airway epithelia with H1N1, H2N2, and H3N2 influenza A virus strains.

Three subtypes of influenza A virus cause human disease: H1N1, H2N2, and H3N2. Although all result in respiratory illness, little is known about how these subtypes infect differentiated airway epithelia. Therefore, we assayed A/PR/8/34 (H1N1), A/Japan/305/57 (H2N2), and X31 (H3N2) influenza virus strains for binding and infection on fully differentiated primary cultures of airway epithelia isolated from human bronchus, grown on semiporous filters at an air-liquid interface. In this model system, viral infectivity was highest when virus was applied to the apical versus the basolateral surface; Japan was most infectious, followed by PR8. The X31 strain showed very low levels of infectivity. Confocal microscopy and fluorescence-resonance energy transfer studies indicated that Japan virus could enter and fuse with cellular membranes, while infection with X31 virions was greatly inhibited. Japan virus could also productively infect human trachea explant tissues. These data show that influenza viruses with SAalpha2,3Gal binding specificity, like Japan, productively infect differentiated human airway epithelia from the apical surface. These data are important to consider in the development of pseudotyped recombinant viral vectors for gene transfer to human airway epithelia for gene therapy.

Discovery of new human beta-defensins using a genomics-based approach.

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.

Synergistic and additive killing by antimicrobial factors found in human airway surface liquid.

Airway surface liquid contains multiple factors thought to provide a first line of defense against bacteria deposited in the airways. Although the antimicrobial action of individual factors has been studied, less is known about how they work in combination. We examined the combined action of six antimicrobial peptides found in airway surface liquid. The paired combinations of lysozyme-lactoferrin, lysozyme-secretory leukocyte protease inhibitor (SLPI), and lactoferrin-SLPI were synergistic. The triple combination of lysozyme, lactoferrin, and SLPI showed even greater synergy. Other combinations involving the human beta-defensins, LL-37, and tobramycin (often administered to cystic fibrosis patients by inhalation) were additive. Because the airway surface liquid salt concentration may be elevated in cystic fibrosis patients, we examined the effect of salt on the synergistic combinations. As the ionic strength increased, synergistic interactions were lost. Our data suggest that the antibacterial potency of airway surface liquid may be significantly increased by synergistic and additive interactions between antimicrobial factors. These results also suggest that increased salt concentrations that may exist in cystic fibrosis could inhibit airway defenses by diminishing these synergistic interactions.

Human coronavirus 229E infects polarized airway epithelia from the apical surface.

Gene transfer to differentiated airway epithelia with existing viral vectors is very inefficient when they are applied to the apical surface. This largely reflects the polarized distribution of receptors on the basolateral surface. To identify new receptor-ligand interactions that might be used to redirect vectors to the apical surface, we investigated the process of infection of airway epithelial cells by human coronavirus 229E (HCoV-229E), a common cause of respiratory tract infections. Using immunohistochemistry, we found the receptor for HCoV-229E (CD13 or aminopeptidase N) localized mainly to the apical surface of airway epithelia. When HCoV-229E was applied to the apical or basolateral surface of well-differentiated primary cultures of human airway epithelia, infection primarily occurred from the apical side. Similar results were noted when the virus was applied to cultured human tracheal explants. Newly synthesized virions were released mainly to the apical side. Thus, HCoV-229E preferentially infects human airway epithelia from the apical surface. The spike glycoprotein that mediates HCoV-229E binding and fusion to CD13 is a candidate for pseudotyping retroviral envelopes or modifying other viral vectors.

Bactericidal activity of mammalian cathelicidin-derived peptides.

Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an alpha-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis.

Increasing epithelial junction permeability enhances gene transfer to airway epithelia In vivo.

Gene transfer to airway epithelia is the most direct approach for treating the progressive lung disease associated with cystic fibrosis. However, the transduction efficiency is poor when viral vectors are applied to the mucosal surface. We reported previously that gene transfer via the apical surface of human airway epithelia in vitro was improved by formulating vectors with ethyleneglycol-bis-(2-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA) in a hypotonic buffer. First, we investigated the mechanism for this enhancement. When 100-nm fluorescent beads were applied to the apical surface in the presence of EGTA, paracellular deposition of the particles was noted. Transmission electron microscopy verified that the epithelial junction complex was disrupted under these conditions. The Ca(2+) chelators EGTA, 1,2-bis (2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ethylenediaminetetraacetic acid all caused a rapid, reversible drop in transepithelial resistance and facilitated gene transfer with retrovirus or adenovirus in vitro. When Ca(2+) chelators were applied to rabbit tracheal epithelia or human nasal epithelia in vivo, the transepithelial voltage decreased, and amiloride sensitivity was lost, suggesting that epithelial junctions opened. Importantly, this novel formulation enhanced both retroviral- and adenoviral-mediated gene transfer to rabbit tracheal epithelia in vivo. This technique may have applications for vector or drug delivery to airway epithelia and other polarized cells.

Development of retroviral vectors for gene transfer to airway epithelia.

Retroviral vectors offer several potential advantages for attaining persistent expression of a therapeutic gene in airway epithelia for diseases such as cystic fibrosis. However, several problems have limited their application. Developments in vector production and the advent of lentiviral vectors have increased the investigation of recombinant retrovirus for gene transfer to airway epithelia. In addition, an improved understanding of some of the barriers limiting gene transfer has led to increased transduction efficiencies. The development of novel vector formulations and the use of new envelope pseudotypes are examples of recent findings that are leading to advances in this field.

KGF and FGF-10 stimulate liquid secretion in human fetal lung.

During fetal life, the pulmonary epithelium secretes liquid that distends the airways and is important for normal lung growth and development. The factors regulating human fetal lung liquid secretion are poorly understood; however, recent studies in murine models show that keratinocyte growth factor (KGF, FGF-7) and fibroblast growth factor 10 (FGF-10) stimulate liquid secretion. We asked whether KGF and FGF-10 stimulate liquid secretion in human fetal lung. First trimester fetal lung explants developed dose-dependent increases in intraluminal volume in response to KGF and FGF-10. Although there were no acute changes in explant transepithelial potential difference in response to KGF (0.1-1000 ng/mL), exposure to 5-50 ng/mL KGF over 60 h depolarized transepithelial potential difference compared with controls. We used ribonuclease protection assays to quantitate the ontogeny and regulation of mRNA expression for KGF and its receptor. Both mRNA were expressed in fetal and postnatal lung. Because the promoter region of the human KGF gene contains cAMP and IL-6 response elements, we asked whether cAMP or IL-6 stimulated expression of KGF or its receptor. We have previously shown that cAMP stimulates liquid secretion in this model. Both cAMP and IL-6 significantly increased expression of KGF but not KGF receptor during a 48-h experiment. Thus, stimulation of liquid secretion in explant models by cAMP may be mediated in part by induction of KGF expression. KGF and FGF-10 may be important paracrine factors regulating liquid secretion in human fetal lung.

Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect.

Several problems limit the application of gene transfer to correct the cystic fibrosis (CF) Cl(-) transport defect in airway epithelia. These include inefficient transduction with vectors applied to the apical surface, a low rate of division by airway epithelial cells, failure of transgene expression to persist, and immune responses to vectors or vector-encoded proteins. To address these issues, we used a feline immunodeficiency virus-based (FIV-based) vector. FIV vector formulated with a calcium chelator transduced fully differentiated, nondividing human airway epithelia when applied to the apical surface. FIV-based vector encoding the cystic fibrosis transmembrane conductance regulator cDNA corrected the Cl(-) transport defect in differentiated CF airway epithelia for the life of the culture (>3 months). When this approach was applied in vivo, FIV vector expressing beta-galactosidase transduced 1-14% of adult rabbit airway epithelia. Transduced cells were present in the conducting airways, bronchioles, and alveoli. Importantly, gene expression persisted, and cells with progenitor capacity were targeted. FIV-based lentiviral vectors may be useful for the treatment of genetic lung diseases such as CF. This article may have been published online in advance of the print edition.