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

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␣), 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␣ 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␣-blocking antibodies, we demonstrated FR␣-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR␣ 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.

Efficient gene delivery to the adult and fetal CNS using pseudotyped non-integrating lentiviral vectors

“…The plasmid expressing the VSVG, pVSVG, has been previously described. 24 pLP-RVG expresses the rabies virus glycoprotein, pHCMVwhvGP64 expresses the baculoviral envelope glycoprotein and was kindly provided by Dr Paul McCray Jr, University of Iowa, and is based on the plasmid previously described by Sinn et al 25 Cell culture and production of pseudotyped NILVs Human fetal kidney 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented Efficient gene delivery to adult and fetal CNS AA Rahim et al with 10% fetal bovine serum and 5% penicillin/streptomycin.…”

Lentiviral transduction of the murine lung provides efficient pseudotype and developmental stage-dependent cell-specific transgene expression

“…14,18 Recent interest has converged on pseudotyping; be it of adeno-associated virus (AAV)-2 genomes with novel AAV serotypes 21,36 or of integrating lentiviral vectors with viral envelopes from lung pathogens. 28,29,[31][32][33]37 The Ebola virus envelope glycoprotein has been successfully used to pseudotype lentivirus and has achieved efficient transduction of the murine lung epithelium and human explants 33,34 although generation of consistently high viral titres has been problematic. Recently the baculovirus gp64 envelope has been shown to produce significant expression in the nasal epithelia of adult mice up to 1 year after application.…”

“…51 The baculovirus envelope protein plasmid pHCMVwhvGP64 was kindly provided by Dr David Parsons and Dr Don Anson, Department of Pulmonary Medicine, Adelaide Women's and Children's Hospital, Australia and is based on that first described by Sinn et al 29 Lentivectors were prepared as follows: producer 293T cells were seeded at 2 Â 10 7 cells per T-150 flask. After 18 h plasmid DNA was mixed in the following amounts per T-150 flask; vector construct (pHR.SINcpptSEW) 40 mg, pMDG.2/ pHCMVwhvGP64 10 mg, pCMVD8.74 30 mg to a final volume of 5 ml in OptiMEM (Invitrogen, Paisley, UK).…”

“…[24][25][26][27] However, airway epithelial gene transfer efficacy with this vector system has previously been restricted by a paucity of pseudotypes, which can be produced at high titres and which can infect lung epithelia from the apical surface. In recent years, apical transduction by lentivirus has been achieved both in vitro and in vivo using a number of viral envelopes from diverse origins, including filovirus, 28,29 baculovirus, 30 influenza 31 and parainfluenza 32 viruses. For example, the Ebola virus envelope glycoprotein has been used successfully to achieve efficient transduction of the murine lung epithelium and human explants 33,34 although generation of consistently high viral titres has been problematic.…”

A bioluminescent imaging mouse model for Marburg virus based on a pseudovirus system

“…1 A major obstacle to research on MARV is the requirement for BSL-4 containment and procedures. 1 The advent of pseudoviruses, which package the GP to the surface of replicationincomplete HIV, 10-13 vesicular stomatitis virus, 14 and feline immunodeficiency virus, 15 allows researchers to safely work with the virus in a BSL-2 environment. In previous MARV studies, the lentiviral NL4-3.Luc.R-E-was the major pseudovirus system used.…”