1075. Goblet Cell Hyperplasia Inhibits Adenovirus but Not AAV5 Mediated Gene Transfer to Differentiated Human Airway Epithelia

AbstractGoblet cell metaplasia and mucus hyper-production are key features of chronic muco-obstructive lung diseases such as asthma, chronic bronchitis, and cystic fibrosis. Various mechanisms lead to goblet cell metaplasia in the airways; the driving mechanism for goblet cell metaplasia in a specific patient may be unknown. We recently found that heat shock protein 90 (HSP90) is important for both IL-13- and IL-17- induced airway goblet cell metaplasia. HSP90 interacts with multiple clients that are important in goblet cell metaplasia including Akt, Jak/STAT, IRS, Notch, and various kinases involved in NFκB signaling. Here, we used a targeted phospho-proteomic approach to identify candidate HSP90 clients modulated by the HSP90-inhibitor geldanamycin. NFκB family members were enriched amongst the top candidate targets of HSP90 inhibition in IL-13 an organotypic model of human airway epithelia. We hypothesized that HSP90 inhibition modulated goblet cell metaplasia by interfering with NFκB signaling. We used transcription factor activation, nuclear translocation, and phospho-specific immunofluorescence assays to investigate how IL-13 exposure and HSP90 inhibition modulated NFκB. We found that HSP90 inhibition prevented goblet cell metaplasia by non-canonically blocking NFκB p100/p52 function in human airway epithelia. NFκB modulation via its interaction with HSP90 is a pharmaceutically feasible therapeutic target for goblet cell metaplasia; this approach may enable treatment of patients with chronic muco-inflammatory lung diseases with both known or unidentified disease-driving mechanisms.

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

Journal of Virology ◽

10.1128/jvi.74.8.3852-3858.2000 ◽

2000 ◽

Vol 74 (8) ◽

pp. 3852-3858 ◽

Cited By ~ 238

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.

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

Journal of Virology ◽

10.1128/jvi.77.10.5902-5910.2003 ◽

2003 ◽

Vol 77 (10) ◽

pp. 5902-5910 ◽

Cited By ~ 87

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.

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

Journal of Virology ◽

10.1128/jvi.73.10.8689-8695.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8689-8695 ◽

Cited By ~ 65

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.

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