[Show abstract][Hide abstract] ABSTRACT: Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase and the receptor for the SARS and NL63 coronaviruses (CoV). Loss of ACE2 function is implicated in severe acute respiratory syndrome (SARS) pathogenesis, but little is known about ACE2 biogenesis and activity in the airways. We report that ACE2 is shed from human airway epithelia, a site of SARS-CoV infection. The regulation of ACE2 release was investigated in polarized human airway epithelia. Constitutive generation of soluble ACE2 was inhibited by DPC 333, implicating a disintegrin and metalloprotease 17 (ADAM17). Phorbol ester, ionomycin, endotoxin, and IL-1beta and TNFalpha acutely induced ACE2 release, further supporting that ADAM17 and ADAM10 regulate ACE2 cleavage. Soluble ACE2 was enzymatically active and partially inhibited virus entry into target cells. We determined that the ACE2 cleavage site resides between amino acid 716 and the putative transmembrane domain starting at amino acid 741. To reveal structural determinants underlying ACE2 release, several mutant and chimeric ACE2 proteins were engineered. Neither the juxtamembrane stalk region, transmembrane domain, nor the cytosolic domain was needed for constitutive ACE2 release. Interestingly, a point mutation in the ACE2 ectodomain, L584A, markedly attenuated shedding. The resultant ACE2-L584A mutant trafficked to the cell membrane and facilitated SARS-CoV entry into target cells, suggesting that the ACE2 ectodomain regulates its release and that residue L584 might be part of a putative sheddase "recognition motif." Thus ACE2 must be cell associated to serve as a CoV receptor and soluble ACE2 might play a role in modifying inflammatory processes at the airway mucosal surface.
[Show abstract][Hide abstract] ABSTRACT: The practical application of gene transfer as a treatment for genetic diseases such as cystic fibrosis or hemophilia has been hindered, in part, by low efficiencies of vector delivery and transgene expression. We demonstrated that a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the envelope glycoprotein from the baculovirus Autographa californica (GP64) efficiently transduces and persistently expresses a reporter gene in respiratory epithelium in the absence of agents that disrupt cellular tight junction integrity. GP64-pseudotyped FIV also efficiently transduced murine hepatocytes after tail vein delivery. To improve the FIV-based vector, we tested the contribution of a series of modifications to luciferase expression in vitro and in vivo. These modifications included the addition of spleen necrosis virus U5 (SNV U5) and mutation of the major splice donor and gag start codon located in the packaging region of the FIV transgene plasmid. After vector modification, we observed significantly enhanced expression of luciferase in respiratory epithelia after nasal application and in the liver after tail vein delivery. In addition, we observed significantly enhanced human factor VIII production after tail vein delivery. These sequential modifications provide an improved FIV lentivirus platform for gene therapy applications and may be applied to other retroviral vectors.
Human Gene Therapy 01/2008; 18(12):1244-52. · 4.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-CoV), resulted in substantial morbidity, mortality, and economic losses during the 2003 epidemic. While SARS-CoV infection has not recurred to a significant extent since 2003, it still remains a potential threat. Understanding of SARS and development of therapeutic approaches have been hampered by the absence of an animal model that mimics the human disease and is reproducible. Here we show that transgenic mice that express the SARS-CoV receptor (human angiotensin-converting enzyme 2 [hACE2]) in airway and other epithelia develop a rapidly lethal infection after intranasal inoculation with a human strain of the virus. Infection begins in airway epithelia, with subsequent alveolar involvement and extrapulmonary virus spread to the brain. Infection results in macrophage and lymphocyte infiltration in the lungs and upregulation of proinflammatory cytokines and chemokines in both the lung and the brain. This model of lethal infection with SARS-CoV should be useful for studies of pathogenesis and for the development of antiviral therapies.
Journal of Virology 02/2007; 81(2):813-21. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2006) 13, S129|[ndash]|S129; doi: 10.1016/j.ymthe.2006.08.396
339. FIV Lentiviral Vector Gene Transfer for Hemophilia A
C.P. Chikkanna-Gowda1, Litao Xie1, Yubin Kang1, Melissa A. Hickey1, Patrick L. Sinn1, Jessica D. Goreham-Voss1, Steven W. Pipe2 and Paul B. McCray Jr.11Program in Gene Therapy, Department of Pediatrics, University of Iowa, Iowa City, IA2Department of Pediatrics, University of Michigan, Ann Arbor, MI
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2006) 13, S270|[ndash]|S270; doi: 10.1016/j.ymthe.2006.08.775
697. Molecular Forms of Feline Immunodeficiency Virus Following Gene Transfer to Airway Epithelia
Marisa Banasik1, Christopher Moressi3, Melissa Hickey2, Todd Scheetz3 and Paul McCray21Genetics PhD Program, University of Iowa, Iowa City, IA2Department of Pediatrics, University of Iowa, Iowa City, IA3Department of Engineering, University of Iowa, Iowa City, IA
[Show abstract][Hide abstract] ABSTRACT: Suboptimal expression from Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) cDNA delivered by viral vectors has, in part, limited the use of gene transfer as a therapy for cystic fibrosis. Previously we described a lentiviral vector developed from feline immunodeficiency virus (FIV) pseudotyped with baculovirus GP64 which transduces nasal epithelia in mice and persistently expresses a reporter gene. The goal of the current project is to further improve transgene expression levels through several modifications to this FIV vector, and to test the optimized vector's effects on protein expression. Modifications include rendering the lentiviral vector “self-inactivating” by deleting the U3 region of the 3’ LTR, thus providing a layer of safety. The FIV central polypurine tract (cPPT) element was inserted upstream of the internal promoter and the woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) was incorporated downstream of the reporter gene. Also, two sites were mutated, a major splice donor and the start codon of the partial Gag sequence, preventing production of undesired product. In addition, a newly identified post- transcriptional control element from the 5’ LTR of spleen necrosis virus (SNVU5) was incorporated into the delivery vector. The Rous sarcoma virus promoter and the reporter gene, firefly luciferase, were used to test relative expression of the intermediate vectors in the production of the optimized FIV vector. Expression was quantified in two model systems transduced with modified FIV lentiviral vector: cultured HT1080 cells and Balb/c mice. As determined by luciferase activity assay, in vitro transductions with intermediate vectors showed an enhanced luciferase expression with wPRE and SNVU5, but no enhanced expression with the addition of cPPT. As determined by CCD camera detection of bioluminescence in vivo, both the wPRE and SNVU5 elements significantly increased luciferase expression in the FIV lentiviral vector, but the cPPT did not. The optimized and intermediate vectors were used to deliver the CFTR gene, and expression was detected by real-time PCR targeting CFTR mRNA. The optimized vector produced approximately 20-fold increase in CFTR mRNA as compared with the control vector. Vector constructs with epithelial specific promoters will be used to transduce airway epithelia in vitro and in vivo, and the contribution of the promoter to expression levels, persistence, and cell specific expression will be assessed. The long-term goal is to use an optimized viral vector construct to express sufficient CFTR protein in the airways of humans with cystic fibrosis to correct the chloride ion transport defect.
[Show abstract][Hide abstract] ABSTRACT: Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.
Journal of Virology 01/2006; 79(23):14614-21. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: TRIM5α is a dominant repressive factor against retrovirus infection. Different species variants of TRIM5α have unique patterns of restriction against lentiviruses and oncoretroviruses. Human TRIM5α (TRIM5αhu) has been shown to effectively inhibit N tropic MLV (N-MLV) but have little effect against human immunodeficiency virus type 1 (HIV-1). Non-human primate species variants of TRIM5α display strong viral restriction against HIV-1. A recent publication (Saenz et al. J Virol., 2005 Dec;79(24):15175-88) detailed the restrictive properties of human and rhesus monkey TRIM5α against FIV when exogenously expressed in the feline cell line CrFK, but saw no restriction in the D17 canine cell line. Using stable mouse cell lines expressing species variants of TRIM5α, we observed no restriction of FIV by TRIM5αhu while both rhesus and African green monkey (AGM) TRIM5α display significant viral inhibition. Additionally, a human TRIM5α chimera containing the restrictive SPRY domain residues from rhesus displayed considerable inhibition of FIV similar to what has been observed with HIV-1. These findings suggest that HIV-1 and FIV capsids pose similar targets for TRIM5α. Ongoing experiments are investigating the restrictive capabilities of endogenous TRIM5αhu on FIV and HIV in several human cell types. Microarray analysis and RT-PCR of several human tissues and cell lines suggest differential expression of TRIM5. The results from this study have implications for the utility of FIV as a gene transfer vector in humans and non-human primates.
[Show abstract][Hide abstract] ABSTRACT: Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.
Journal of Virology 11/2005; 79(20):12818-27. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hemophilia A is a clinically important coagulation disorder caused by the lack or abnormality of plasma coagulation factor VIII (FVIII). Gene transfer of the FVIII cDNA to hepatocytes using lentiviral vectors is a potential therapeutic approach. We investigated the efficacy of feline immunodeficiency virus (FIV)-based vectors in targeting hepatocytes and correcting FVIII deficiency in a hemophilia A mouse model. Several viral envelope glycoproteins were screened for efficient FIV vector pseudotyping and hepatocyte transduction. The GP64 glycoprotein from baculovirus Autographa californica multinuclear polyhedrosis virus pseudo-typed FIV efficiently and showed excellent hepatocyte tropism. The GP64-pseudotyped vector was stable in the presence of human or mouse complement. Inclusion of a hybrid liver-specific promoter (murine albumin enhancer/human alpha1-antitrypsin promoter) further enhanced transgene expression in hepatocytes. We generated a GP64-pseudotyped FIV vector encoding the B domain-deleted human FVIII coding region driven by the liver-specific promoter, with 2 beneficial point mutations in the A1 domain. Intravenous vector administration conferred sustained FVIII expression in hemophilia A mice for several months without the generation of anti-human FVIII antibodies and resulted in partial phenotypic correction. These findings demonstrate the utility of GP64-pseudotyped FIV lentiviral vectors for targeting hepatocytes to correct disorders associated with deficiencies of secreted proteins.
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2005) 11, S1|[ndash]|S1; doi: 10.1016/j.ymthe.2005.06.004
1. Persistent Gene Expression in Mouse Nasal Epithelia Following Baculovirus GP64 Pseudotyped FIV-Based Gene Transfer
Patrick L. Sinn1, Erin R. Burnight1, Melissa A. Hickey1, Gary W. Blissard2 and Paul B. McCray Jr11Pediatrics, Program in Gene Therapy, The University of Iowa, Iowa City, IA2Boyce Thompson Institute, Cornell University, Ithaca, NY
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2005) 11, S232|[ndash]|S232; doi: 10.1016/j.ymthe.2005.07.141
601. Persistent Expression of Factor VIII In Vivo Following FIV Lentiviral Gene Transfer
Yubin Kang1, Litao Xie1, Diane Thi Tran1, Colleen S. Stein2, Melissa Hickey1, Beverly L. Davidson2 and Paul B. McCray11Pediatrics, University of Iowa, Carver College of Medicine, Iowa City, IA2Internal Medicine, University of Iowa, College of Medicine, Iowa City, IA
[Show abstract][Hide abstract] ABSTRACT: A feline immunodeficiency virus (FIV)-based lentiviral vector was pseudotyped to identify envelope (env) glycoproteins that direct efficient gene transfer to pulmonary epithelia for the treatment or prevention of lung diseases. The envelope glycoprotein from the Jaagsiekte sheep retrovirus (JSRV) is a candidate under investigation. We utilized high titer FIV vector (>10(8) TU/ml) pseudotyped with the JSRV env glycoprotein (JSRVFIV) to study the transduction of polarized primary cultures of human airway epithelia and receptor/vector interactions. The reported receptor for JSRV, hyaluronidase 2 (HYAL2), is a GPI-linked protein. We expressed FLAG-tagged HYAL2 in polarized airway epithelia using an adenoviral vector and documented that the HYAL2 protein sorts predominantly to the apical surface. Of interest, the efficiency of gene transfer with apically applied JSRV-FIV was markedly less than FIV pseudotyped with VSV-G, even in Ad-HYAL2 complemented epithelia. The inefficient gene transfer with JSRV-FIV in HYAL2 complemented cells suggests that factors other than receptor abundance limit apical gene transfer efficiency with this envelope. JSRV-FIV transduced the distal lung epithelia of rabbits in vivo and transduced primary cultures of rabbit type II cells with 100-fold greater efficiency than primary cultures of rabbit tracheal cells. These data indicate that a lentivirus pseudotyped with the JSRV envelope glycoprotein transduces type II cells with greater efficiency than conducting airway epithelia and provides an example of glycoprotein-mediated cell-specific tropism within a tissue with a widely heterogeneous cell population.
Human Gene Therapy 04/2005; 16(4):479-88. · 4.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lentiviral vectors mediate long term transgene expression through integration into host genomic DNA. However, integration comes with potential risks of insertional mutagenesis. In cases where transient gene expression is desired, an integrase (IN) deficient retroviral vector might be a useful alternative. Furthermore, IN deficient vectors may provide some advantages over alternative methods of transient gene expression due to the ability to pseudotype these vectors with heterologous envelopes that target specific cell types. Class I IN mutants have point mutations in the catalytic triad amino acids (DX39-58 DX35 E) of IN that disable this protein but leave other viral proteins functional. Transient gene expression from IN deficient vectors is mediated from episomal forms of vector DNA. 1-LTR and 2-LTR circles form through homologous recombination and non-homologous end joining, respectively, of the linear form of the reverse transcribed viral DNA. Although 1-LTR and 2-LTR circles are unable to efficiently integrate in the presence of functional IN protein, they can mediate transgene expression. It was previously reported that IN deficient HIV and FIV vectors could mediate transient gene transfer. In growth arrested cells, transgene expression was maintained. However, in rapidly dividing cells transgene expression was lost, and duration of expression was related to the rate of cell division. Because some gene transfer targets are mitotically quiescent and slowly dividing cells, IN deficient mutants may promote long term, though ultimately transient, gene expression. We hypothesized that IN deficient FIV vectors can be used to maintain transient gene expression in slowly dividing cell types, such as airway epithelia. We developed IN deficient FIV vectors with single (D66V, D118A, E154G), double (D66V/D118A, D66V/E154G, D118A/E154G), and triple (D66V/D118A/E154G) mutations that disrupt the catalytic triad of the IN protein. Preliminary data indicate that the FIV D66V IN mutant has titers similar to wild type IN vectors. Furthermore, we documented the production of 1-LTR and 2-LTR circles in human cell line cultures transduced with FIV D66V IN deficient vector. Ongoing studies are comparing the persistence of transgene expression in IN deficient versus wild type IN FIV in dividing and non-dividing cells. Furthermore, to ensure that transgene expression seen in IN deficient vectors represents episomal and not integrated vector DNA, Southern blotting will be used. IN deficient lentiviral vectors may have applications for transient gene expression, long term expression in slowly dividing cells, and can be used as controls in studies with integration competent vectors.
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2004) 9, S65–S65; doi: 10.1016/j.ymthe.2004.06.131
171. Enhancing hepatocyte gene transfer with baculovirus GP64 Glycoprotein*
Yubin Kang1, Litao Xie1, Diane Thi Tran1, Melissa Hickey1, Colleen S. Stein2, Beverly L. Davidson2 and Paul B. McCray Jr11Program in Gene Therapy, Department of Pediatrics2Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA*Supported by the Career Development Award from the National Hemophilia Foundation
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2004) 9, S261–S261; doi: 10.1016/j.ymthe.2004.06.587
688. Pseudotyping FIV-based lentiviral vectors with baculovirus GP64 confers apical entry into airway epithelia
Patrick L. Sinn1, Erin R. Burnight1, Doug E. Dylla2, Christina S. Firm2, Melissa A. Hickey1, Gary W. Blissard3 and Paul B. McCray Jr11Pediatrics, Program in Gene Therapy, The University of Iowa, Iowa City, IA2Genetics, The University of Iowa, Iowa City, IA3Cornell University, Ithaca, NY
[Show abstract][Hide abstract] ABSTRACT: Gene transfer to the airway epithelia can correct the ion transport and host defense defects associated with cystic fibrosis (CF). However, the clinical application of gene therapy for CF is limited by several problems including adverse immune responses, inefficient transduction, and the failure of gene expression to persist. We hypothesized that the transduction efficiency of a (feline immunodeficiency virus) FIV-based lentivirus for the apical surface of polarized human airway epithelia (HAE) could be improved by pseudotyping the vector with glycoproteins from non-retroviral enveloped viruses. We found that glycoproteins from the arenavirus lymphocytic choriomeningitis virus (LCMV, WE54 strain) produced FIV titers similar to VSV-G FIV (108-109 TU/ml). Application of FIV pseudotyped with LCMV to well differentiated HAE yielded significantly more efficient gene transfer from the apical than the basolateral surface. Transduction by LCMV FIV from the apical surface was 3–4 fold more efficient than VSV-G FIV from the basolateral surface. Additionally, transduction was decreased by inhibitors of endosomal acidification, consistent with entry via a pH-sensitive endosomal pathway. Alpha-dystroglycan has been identified as a high affinity receptor for some arenavirus family members, including the WE54 strain of LCMV. By immunohistochemistry, alpha-dystroglycan was abundantly expressed on both the apical and basolateral surfaces of airway epithelia. Pretreatment of epithelia with laminin or antibodies to alpha-dystroglycan partially inhibited gene transfer with LCMV-FIV, suggesting that alpha-dystroglycan serves as a receptor in HAE. Vectors with a single point mutation to the LCMV WE54 envelope, designed to generate LCMV FIV virions with low affinity to alpha-dystroglycan, transduced airway epithelia 5–12 fold more efficiently from the apical surface than the high affinity WE54 glycoprotein pseudotyped virions. Transduction by the modified pseudotype was not inhibited by laminin or alpha-dystroglycan blocking antibody. Transduction levels with the low affinity alpha-dystroglycan binding envelope were also increased following basolateral application. These results with an envelope with low affinity for alpha-dystroglycan suggest that there is at least one additional unidentified viral receptor or cofactor for LCMV binding in airway epithelia. LCMV pseudotyped FIV provides a new tool to deliver transgenes to polarized airway epithelia via the apical surface.
[Show abstract][Hide abstract] 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 (FR alpha), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR alpha 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 FR alpha-blocking antibodies, we demonstrated FR alpha-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR alpha 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.
Journal of Virology 06/2003; 77(10):5902-10. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.