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

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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Incorporation of Adeno-Associated Virus in a Calcium Phosphate Coprecipitate Improves Gene Transfer to Airway Epithelia In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.74.1.535-540.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 535-540 ◽

Cited By ~ 27

Author(s):

Robert W. Walters ◽

Dongsheng Duan ◽

John F. Engelhardt ◽

Michael J. Welsh

Keyword(s):

Calcium Phosphate ◽

Gene Transfer ◽

Mouse Lung ◽

Apical Surface ◽

Airway Epithelia ◽

Adeno Associated Virus ◽

Human Airway

ABSTRACT Adeno-associated virus (AAV) is inefficient at infecting differentiated airway epithelia because of a lack of receptors at the apical surface. We hypothesized that incorporation of AAV in a calcium phosphate coprecipitate would circumvent this barrier. Interestingly, coprecipitation of AAV type 2 improved gene transfer to differentiated human airway epithelia in vitro and to the mouse lung in vivo. These results suggest that delivery of AAV as a CaPicoprecipitate may significantly enhance its utility for gene transfer to the airway epithelia in vivo.

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756. Distinct Classes of Proteasome-Modulating Agents Cooperatively Augment Recombinant Adeno-Associated Virus Type 2 and Type 5-Mediated Transduction from the Apical Surface of Human Airway Epithelia

Molecular Therapy ◽

10.1016/j.ymthe.2004.06.689 ◽

2004 ◽

Vol 9 ◽

pp. S287-S288

Keyword(s):

Virus Type ◽

Apical Surface ◽

Airway Epithelia ◽

Adeno Associated Virus ◽

Human Airway ◽

Human Airway Epithelia

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Distinct Classes of Proteasome-Modulating Agents Cooperatively Augment Recombinant Adeno-Associated Virus Type 2 and Type 5-Mediated Transduction from the Apical Surfaces of Human Airway Epithelia

Journal of Virology ◽

10.1128/jvi.78.6.2863-2874.2004 ◽

2004 ◽

Vol 78 (6) ◽

pp. 2863-2874 ◽

Cited By ~ 88

Author(s):

Ziying Yan ◽

Roman Zak ◽

Yulong Zhang ◽

Wei Ding ◽

Simon Godwin ◽

...

Keyword(s):

Cell Lines ◽

Virus Type ◽

Cell Types ◽

Nuclear Accumulation ◽

Cell Type Specificity ◽

Airway Epithelia ◽

Adeno Associated Virus ◽

Human Airway ◽

Human Airway Epithelia

ABSTRACT Tripeptidyl aldehyde proteasome inhibitors have been shown to effectively increase viral capsid ubiquitination and transduction of recombinant adeno-associated virus type 2 (rAAV-2) and rAAV-5 serotypes. In the present study we have characterized a second class of proteasome-modulating agents (anthracycline derivatives) for their ability to induce rAAV transduction. The anthracycline derivatives doxorubicin and aclarubicin were chosen for analysis because they have been shown to interact with the proteasome through a mechanism distinct from that of tripeptidyl aldehydes. Our studies demonstrated that doxorubicin and aclarubicin also significantly augmented rAAV transduction in airway cell lines, polarized human airway epithelia, and mouse lungs. Both tripeptidyl aldehyde and anthracycline proteasome-modulating agents similarly augmented nuclear accumulation of rAAV in A549 and IB3 airway cell lines. However, these two cell types demonstrated cell specificity in the ability of N-acetyl-l-leucyl-l-leucyl-l-norleucine (LLnL) or doxorubicin to augment rAAV transduction. Interestingly, the combined administration of LLnL and doxorubicin resulted in substantially increased transduction (>2,000-fold) following apical infection of human polarized epithelia with either rAAV-2 or rAAV-5. In summary, the cell type specificity of LLnL and doxorubicin to induce rAAV transduction, together with the ability of these compounds to synergistically enhance rAAV transduction in polarized airway epithelial induction, suggests that these two classes of compounds likely modulate different proteasome functions that affect rAAV transduction. Findings from this study provide new insights into how modulation of proteasome function can be effectively used to augment rAAV transduction in airway epithelia for gene therapy of cystic fibrosis.

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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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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

Journal of Virology ◽

10.1128/jvi.75.16.7703-7711.2001 ◽

2001 ◽

Vol 75 (16) ◽

pp. 7703-7711 ◽

Cited By ~ 34

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.

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