scholarly journals Lentivirus Vectors Pseudotyped with Filoviral Envelope Glycoproteins Transduce Airway Epithelia from the Apical Surface Independently of Folate Receptor Alpha

2003 ◽  
Vol 77 (10) ◽  
pp. 5902-5910 ◽  
Author(s):  
Patrick L. Sinn ◽  
Melissa A. Hickey ◽  
Patrick D. Staber ◽  
Douglas E. Dylla ◽  
Scott A. Jeffers ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Folate Receptor ◽  
Primary Cultures ◽  
Apical Surface ◽  
Airway Epithelia ◽  
Gene Transfer Efficiency ◽  
Human Airway ◽  
Human Airway Epithelia

ABSTRACT The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FRα), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FRα on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FRα-blocking antibodies, we demonstrated FRα-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FRα was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.

scholarly journals Apical Localization of the Coxsackie-Adenovirus Receptor by Glycosyl-Phosphatidylinositol Modification Is Sufficient for Adenovirus-Mediated Gene Transfer through the Apical Surface of Human Airway Epithelia

2001 ◽  
Vol 75 (16) ◽  
pp. 7703-7711 ◽  
Author(s):  
Robert W. Walters ◽  
Wouter van't Hof ◽  
Su Min P. Yi ◽  
Mary K. Schroth ◽  
Joseph Zabner ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Transfer ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Wild Type ◽  
Airway Epithelia ◽  
Glycosyl Phosphatidylinositol ◽  
Human Airway ◽  
Human Airway Epithelia

ABSTRACT In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl− transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis.

scholarly journals Bronchoalveolar Fluid Is Not a Major Hindrance to Virus-Mediated Gene Therapy in Cystic Fibrosis

2002 ◽  
Vol 76 (20) ◽  
pp. 10437-10443 ◽  
Author(s):  
C. P. Rooney ◽  
G. M. Denning ◽  
B. P. Davis ◽  
D. M. Flaherty ◽  
J. A. Chiorini ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Airway Epithelium ◽  
Transfer Efficiency ◽  
Apical Surface ◽  
Coxsackie Adenovirus Receptor ◽  
Gene Transfer Efficiency ◽  
Human Airway Epithelium ◽  
Bronchoalveolar Fluid

ABSTRACT Successfully targeting the airway epithelium is essential for gene therapy of some pulmonary diseases. However, the airway epithelium is resistant to virus-mediated gene transfer with commonly used vectors. Vectors that interact with endogenously expressed receptors on the apical surface significantly increase gene transfer efficiency. However, other endogenous components involved in host immunity may hinder virus-mediated gene transfer. We tested the effect of bronchoalveolar lavage liquid (BAL) from patients with cystic fibrosis (CF), BAL from subjects without CF (non-CF BAL), Pseudomonas aeruginosa-derived proteins, and an array of inflammatory proteins on gene transfer mediated by adeno-associated virus type 5 (AAV5) and adenovirus targeted to an apically expressed glycosylphosphatidylinositol-modified coxsackie-adenovirus receptor. We found that neither CF BAL nor its components had a significant effect on gene transfer to human airway epithelium by these vectors. Non-CF BAL significantly impaired adenovirus-mediated gene transfer. Removal of immunoglobulins in non-CF BAL restored gene transfer efficiency. As virus vectors are improved and mechanisms of humoral immunity are elucidated, barriers to successful gene therapy found in the complex environment of the human lung can be circumvented.

scholarly journals Adeno-Associated Virus Type 5 (AAV5) but Not AAV2 Binds to the Apical Surfaces of Airway Epithelia and Facilitates Gene Transfer

2000 ◽  
Vol 74 (8) ◽  
pp. 3852-3858 ◽  
Author(s):  
Joseph Zabner ◽  
Michael Seiler ◽  
Robert Walters ◽  
Robert M. Kotin ◽  
Wendy Fulgeras ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Virus Type ◽  
Target Cells ◽  
Apical Surface ◽  
Airway Epithelia ◽  
Adeno Associated Virus ◽  
Human Airway ◽  
Human Airway Epithelia

ABSTRACT In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring β-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5-derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2.

Basolateral chloride current in human airway epithelia

2007 ◽  
Vol 293 (4) ◽  
pp. L991-L999 ◽  
Author(s):  
Omar A. Itani ◽  
Fred S. Lamb ◽  
James E. Melvin ◽  
Michael J. Welsh
Keyword(s):  
Cystic Fibrosis ◽  
Signaling Pathways ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Airway Epithelia ◽  
Human Airway ◽  
Well Differentiated ◽  
Human Airway Epithelia

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl− absorption. However, the pathways for basolateral Cl− exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl− conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl− conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl− current that was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl− > Br− >> I−. Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca2+ and inducing cell swelling had no effect. The basolateral Cl− current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl− currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl− conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl− conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl− movement.

scholarly journals A Chimeric Type 2 Adenovirus Vector with a Type 17 Fiber Enhances Gene Transfer to Human Airway Epithelia

1999 ◽  
Vol 73 (10) ◽  
pp. 8689-8695 ◽  
Author(s):  
Joseph Zabner ◽  
Miguel Chillon ◽  
Teresa Grunst ◽  
Thomas O. Moninger ◽  
Beverly L. Davidson ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Transfer ◽  
Recombinant Adenovirus ◽  
Adenovirus Vector ◽  
Adenovirus Type ◽  
Wild Type ◽  
Airway Epithelia ◽  
Human Airway ◽  
Human Airway Epithelia

ABSTRACT In studies of the genetic disease cystic fibrosis, recombinant adenovirus type 2 (Ad2) and Ad5 are being investigated as vectors to transfer cystic fibrosis transmembrane conductance regulator cDNA to airway epithelia. However, earlier work has shown that human airway epithelia are resistant to infection by Ad2 and Ad5. Therefore, we examined the efficiency of other adenovirus serotypes at infecting airway epithelia. We found that several serotypes of adenoviruses, in particular, wild-type Ad17, infected a greater number of cells than wild-type Ad2. The increased efficiency of wild-type Ad17 could be explained by increased fiber-dependent binding to the epithelia. Therefore, we constructed a chimeric virus, Ad2(17f)/βGal-2, which is identical to Ad2/βGal-2 with the exception of having the fiber protein of Ad17 replace Ad2 fiber. This vector retained the increased binding and efficiency of gene transfer to well-differentiated human airway epithelia. These data suggest that inclusion of Ad17 fiber into adenovirus vectors may improve the outlook for gene delivery to human airway epithelia.

scholarly journals Human Coronavirus 229E Infects Polarized Airway Epithelia from the Apical Surface

2000 ◽  
Vol 74 (19) ◽  
pp. 9234-9239 ◽  
Author(s):  
Guoshun Wang ◽  
Camille Deering ◽  
Michael Macke ◽  
Jianqiang Shao ◽  
Royce Burns ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Primary Cultures ◽  
Apical Surface ◽  
Human Coronavirus ◽  
Airway Epithelia ◽  
Human Airway ◽  
Apical Side ◽  
Tract Infections ◽  
Human Coronavirus 229E ◽  
Human Airway Epithelia

ABSTRACT 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.

scholarly journals Measles Virus Preferentially Transduces the Basolateral Surface of Well-Differentiated Human Airway Epithelia

2002 ◽  
Vol 76 (5) ◽  
pp. 2403-2409 ◽  
Author(s):  
Patrick L. Sinn ◽  
Greg Williams ◽  
Sompong Vongpunsawad ◽  
Roberto Cattaneo ◽  
Paul B. McCray
Keyword(s):  
Vaccine Strain ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
Primary Cultures ◽  
Egfp Expression ◽  
Apical Surface ◽  
Airway Epithelia ◽  
Human Airway ◽  
Well Differentiated ◽  
Human Airway Epithelia

ABSTRACT Measles virus (MV) is typically spread by aerosol droplets and enters via the respiratory tract. The progression of MV infection has been widely studied; yet, the pathway for virus entry in polarized human airway epithelia has not been investigated. Herein we report the use of a replication-competent Edmonston vaccine strain of MV expressing enhanced green fluorescent protein (MV-eGFP) to infect primary cultures of well-differentiated human airway epithelia. Previous studies with polarized Caco-2 cells (intestine-derived human epithelia) and MDCK cells (kidney-derived canine epithelia) demonstrated that MV primarily infected and exited the apical surface. In striking contrast, our results indicate that MV preferentially transduces human airway cells from the basolateral surface; however, virus release remains in an apical direction. When MV-eGFP was applied apically or basolaterally to primary cultures of airway epithelia, discrete foci of eGFP expression appeared and grew; however, the cell layer integrity was maintained for the duration of the study (7 days). Interestingly, utilizing immunohistochemistry and confocal microscopy, we observed widespread expression of the receptor for the vaccine strain of MV (CD46) at greatest abundance on the apical surface of the differentiated human airway epithelia as well as in human tracheal tissue sections. These data suggest that the progression of MV infection through the respiratory epithelium may involve pathways other than direct binding and entry through the apical surface of airway epithelia.

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