491. Improved Apical Airway Epithelia Targeting Properties of FIV Vector Pseudotyped with Envelopes from LCMV

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.