Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo

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Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo Gary P. Kobinger1†, Daniel J. Weiner1,2†, Qian-Chun Yu1, and James M. Wilson1*

Traditional gene therapy vectors have demonstrated limited utility for treatment of chronic lung diseases such as cystic fibrosis (CF). Herein we describe a vector based on a Filovirus envelope protein-pseudotyped HIV vector, which we chose after systematically evaluating multiple strategies. The vector efficiently transduces intact airway epithelium from the apical surface, as demonstrated in both in vitro and in vivo model systems. This shows the potential of pseudotyping in expanding the utility of lentiviral vectors. Pseudotyped lentiviral vectors may hold promise for the treatment of CF.

Cystic fibrosis (CF) is caused by functional absence of the CF transmembrane conductance regulator (CFTR) protein1. Successful gene replacement therapy for CF has been limited by the lack of a suitable vector that achieves good transduction of airway epithelia. Poor transduction efficiency, short duration of expression, an exuberant inflammatory response, or combinations of these problems hamper existing vectors such as adenovirus or adeno-associated virus serotype 2 (AAV-2). Advantages of lentiviral vectors include transduction of nondividing cells, sustained transgene expression from the integrated provirus, and simplicity in modifying tropism2. Many unsuccessful attempts have been made to biochemically alter the envelope protein that is packaged on retroviral vectors such as the amphotropic envelope of the murine leukemia virus (MuLV)3,4 in order to modify the vector tropism. Exploiting the ability of retroviral vectors to incorporate envelope proteins from different viruses onto their viral membrane (i.e., pseudotyping) may be useful. Pseudotyping may allow for efficient gene transfer into specific tissues via a particular cellular domain; indeed, the viral envelope contributes to the tropism of the virus and to whether a virus–host cell interaction will occur. Initial studies with lentiviral vector pseudotyped with vesicular stomatitis virus G (VSV-G) envelope for transferring genes to differentiated airway epithelia have been disappointing because of limited entry5. VSV-G-pseudotyped retroviral vector was shown to transduce airway epithelia in vivo only when access to the basolateral side is provided by opening tight junctions using agents that may not be practical for clinical application6,7. The aim of this study is to identify a viral envelope protein that can be packaged with an HIV-based vector, which would allow for efficient and nontoxic transduction of airway epithelia. Pseudotyping HIV vectors with the envelope from the Zaire strain of the Ebola virus (EboZ) resulted in efficient transduction of airway epithelium in vitro and in vivo. Filoviridae, including Ebola and Marburg viruses, are enveloped, nonsegmented, negative-sense RNA viruses that are pleomorphic and appear as nonfilamentous forms. Our data demonstrate the combined benefits of lentiviruses (transduction of nondividing cells and possibility for stable expression)

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and Filoviridae envelope proteins (tropism for respiratory epithelium). With such envelope pseudotyping, lentiviral vectors may hold promise for treatment of airway diseases such as CF.

Results Creation of pseudotyped lentiviral vectors. The following viral envelopes were used for pseudotyping: MuLV amphotropic envelope, Mokola envelope, EboZ envelope, Ebola-Reston (EboR) envelope, influenza-hemagglutinin (HA) envelope, and respiratory syncytial virus (RSV) F and G envelope proteins. All pseudotyped viruses were produced in parallel under the same conditions for every experiment. Because each viral envelope protein used to pseudotype vector conferred specific tropism to the vector, titers established by limiting dilution on target cell lines were different and thus not used for normalizing the amount of input vector (Table 1). Consequently, each transduction was performed by using the same volume of concentrated vector produced from the same amount of cells transfected under the same conditions. Stocks were assayed for reverse transcriptase (RT) activity as described8; viral stocks had an average activity of 2 × 105 counts/min/µl. Pseudotyped vectors applied on tissues for transduction demonstrated similar RT activity, indicating that comparable amounts of viruslike particles were used (data not shown). Identification of viral envelopes that mediate apical transduction of human airway. Viruses pseudotyped with a variety of envelopes were applied to air/liquid interface (ALI) cultures of airway epithelial cells apically or basolaterally and analyzed four days later for green fluorescent protein (GFP) expression. Partially concentrated virus (50 µl, concentrated 100-fold) was added to the apical or basolateral side of ALI cultures. After basolateral application, the panel of pseudotyped HIV vectors transduced between 0 and 110 cells/cm2 (Fig. 1A). In contrast, apical application resulted in poor transduction by all pseudotyped vectors with the exception of EboZpseudotyped virus, for which >200 positive cells/cm2 were detected. These experiments demonstrated the relative efficiency of EboZpseudotyped virus compared to other pseudotyped vectors. However, the overall transduction efficiency is 80% of β-gal-expressing cells after 28 days. On average, 30% of the entire tracheal epithelium was transduced by EboZ-pseudotyped HIV vector at day 28 and 24% at day 63. Interestingly, high expression was observed in submucosal glands (an average of 65% of cells) of airways from animals receiving EboZ-pseudotyped HIV vector. No submucosal gland staining was seen in control animals receiving vehicle, and 500 Ω/cm2) was generated, and no defects in the membrane could be visualized using light microscopy. For screening of viral envelopes, ALI cultures were transduced with GFP-encoding viruses applied from the apical or basolateral side. Transepithelial resistance was measured 24 h after infection and remained >500 Ω/cm2 (data not shown), indicating that the epithelial integrity was not compromised. Cultures were examined using fluorescent microscopy at four days after transduction, and GFP-expressing cells were counted by examining 20 fields at 100× magnification and extrapolating for the surface area of the support. Tracheal explant. Small pieces (0.5 cm2) were excised from explanted normal or CF human airways and placed on collagen-coated permeable supports. Tissue could be fed from the basolateral surface with medium as above. Tissues were infected with 50–100 µl of viral stocks from the apical surface and incubated for 2–4 h. Viral titers were determined by limiting dilution on 293T cells, demonstrating 1 × 107 GFP-transducing units (TU)/ml for EboZHIV and 1 × 109 TU/ml for VSV-G-HIV vector. Medium was replaced and the tissue was then submerged in medium overnight, with replacement every 12 h. Tissue was fixed in 0.5% glutaraldehyde, stained with X-gal at 37°C for 3–12 h, and processed for paraffin embedding.

Animal models. C57BL/6 mice (six to eight weeks of age) were anesthetized using intraperitoneal ketamine/xylazine. Using standard techniques, the trachea was exposed through a midline incision, 100 µl of vector preparation were instilled using a syringe, and the subcutaneous tissues were sutured closed. Viral titers were determined by limiting dilution on 293T cells, demonstrating 5 × 107–5 × 108 TU/ml for EboZ-HIV and 5 × 109–5 × 1010 TU/ml for VSV-G-HIV vector. Animals were maintained in the animal facility until necropsy. At necropsy, the lungs were inflated with OCT/PBS (1:1) and processed using cryofixation. Cryosections (10 µm) were prepared and

DNA construction and virus production. The helper packaging construct pCMV∆R8.2, encoding for the HIV helper function, the transfer vector pHR′LacZ encoding for the β-gal, and plasmids encoding for envelope proteins were used for triple transfection. The transfer vector pHR′EGFP was generated by cloning the BamHI/blunted BclI containing the EGFP open

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stained with X-gal overnight. Transduction efficiency was estimated by examining 20–25 high-power fields from 16 cryosections spaced throughout the tissue block (at 400 µm intervals). Two animals were treated with each pseudotyped virus, and experiments were duplicated. All animal procedures were approved by the Wistar Institute Institutional Animal Care and Use Committee.

ed, and refixed with 2.5% glutaraldehyde. After osmication, the cells were processed for transmission electron microscopy as above.

Acknowledgments The authors thank Dr. Inder Verma for providing pCMV∆R8.2, pHR′LacZ, and the VSV-G envelope expressor, Dr. Paul Bates for providing the EboZ and EboR envelope plasmids as well as antibodies against the Ebola envelope glycoprotein, Dr. Eric Hunter for providing the BH-RCANsHA envelope plasmid, Dr. Jacob Reizer for providing the Mokola envelope plasmid, Dr. Eric Cohen for providing the SVCMV in plasmid and the amphotropic MuLV envelope, Dr. Christian Moser for insightful discussion, and Dr. John Tazelaar for assistance with tissue processing and microscopy. G.P.K. is the recipient of a fellowship from the Medical Research Council of Canada. This work was funded by grants from the National Institutes of Health (DK47757-08), the CF Foundation, and Genovo, Inc., a biotechnology company Dr. Wilson founded and in which he has equity.

Electron microscopy. 293T cells were triple-transfected by calcium phosphate as described above. After 64 h, cells were fixed in 2.5% glutaraldehyde in PBS and postfixed in 1% osmium tetroxide. Cells were then enclosed in 1% agar, treated with 1% uranyl acetate, and embedded in Epon. Ultrathinsection specimens were analyzed with a Phillips transmission electron microscope at a voltage of 80 kV. For pre-embedding immunolabeling of Ebola virus envelope glycoprotein, transfected 293T cells were collected 72 h after transfection, washed, and fixed with 2% paraformadehyde and 0.05% glutaraldehyde. After washing, the cells were incubated with antibody against the EboZ envelope glycoprotein, then with 15 nm gold-conjugated secondary antibody. At the end of incubation, the cells were washed several times, pellet-

Received 1 August 2000; accepted 20 December 2000

3155–3160 (1998). 18. Chan, S., Speck, R., Ma, M. & Goldsmith, M. Distinct mechanisms of entry by envelope glycoproteins of Marburg and Ebola (Zaire) viruses. J. Virol. 74, 4933–4937 (2000). 19. Bals, R. et al. Transduction of well-differentiated airway epithelium by recombinant adeno-associated virus is limited by vector entry. J. Virol. 73, 6085–6088 (1999). 20. Engelhardt, J.F., Yankaskas, J.R. & Wilson, J.M. In vivo retroviral gene transfer into human bronchial epithelia of xenografts. J. Clin. Invest. 90, 2598–2607 (1992). 21. Olsen, J.C. et al. Correction of the apical membrane chloride permeability defect in polarized cystic fibrosis airway epithelia following retroviral-mediated gene transfer. Hum. Gene Ther. 3, 253–266 (1992). 22. Zabner, J. et al. Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J. Virol. 74, 3852–3858 (2000). 23. Yonemitsu, Y. et al. Efficient gene transfer to airway epithelium using recombinant Sendai virus. Nat. Biotechnol. 18, 970–973 (2000). 24. Lopez-Vidriero, M.T. & Reid, L. Chemical markers of mucous and serum glycoproteins and their relation to viscosity in mucoid and purulent sputum from various hypersecretory diseases. Am. Rev. Respir. Dis. 117, 465–477 (1978). 25. Crawford, I. et al. Immunocytochemical localization of the cystic fibrosis gene product CFTR. Proc. Natl. Acad. Sci. USA 88, 9262–9266 (1991). 26. Duan, D., Yue, Y., Yan, Z., Yang, J. & Engelhardt, J.F. Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus. J. Clin. Invest. 105, 1573–1587 (2000). 27. Wang, G. et al. Influence of cell polarity on retrovirus-mediated gene transfer to differentiated human airway epithelia. J. Virol. 72, 9818–9826 (1998). 28. Engelhardt, J.F. et al. Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1-deleted adenoviruses. Nat. Genet. 4, 27–34 (1993). 29. Blomer, U. et al. Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J. Virol. 71, 6641–6649 (1997). 30. Lodge, R., Subbramanian, R.A., Forget, J., Lemay, G. & Cohen, E.A. MuLV-based vectors pseudotyped with truncated HIV glycoproteins mediate specific gene transfer in CD4+ peripheral blood lymphocytes. Gene Ther. 5, 655–664 (1998). 31. Yao, X.J., Kobinger, G., Dandache, S., Rougeau, N. & Cohen, E. HIV-1 Vpr-chloramphenicol acetyltransferase fusion proteins: sequence requirement for virion incorporation and analysis of antiviral effect. Gene Ther. 6, 1590–1599 (1999). 32. Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471 (1998).

1. Davis, P.B., Drumm, M. & Konstan, M.W. Cystic fibrosis. Am. J. Respir. Crit. Care Med. 154, 1229–1256 (1996). 2. Buchschacher, G.L. Jr. & Wong-Staal, F. Development of lentiviral vectors for gene therapy for human diseases. Blood. 95, 2499–2504 (2000). 3. Peng, K.W. & Russell, S. J. Viral vector targeting. Curr. Opin. Biotechnol. 10, 454–457 (1999). 4. Schnierle, B.S. & Groner, B. Retroviral targeted delivery. Gene Ther. 3, 1069–1073 (1996). 5. Goldman, M.J., Lee, P.S., Yang, J.S. & Wilson, J.M. Lentiviral vectors for gene therapy of cystic fibrosis. Hum Gene Ther. 8, 2261–2268 (1997). 6. Wang, G. et al. Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect. J Clin. Invest. 104, R55–62 (1999). 7. Johnson, L.G., Olsen, J.C., Naldini, L. & Boucher, R.C. Pseudotyped human lentiviral vector-mediated gene transfer to airway epithelia in vivo. Gene Ther. 7, 568–574 (2000). 8. Kobinger, G.P. et al. Virion-targeted viral inactivation of human immunodeficiency virus type 1 by using Vpr fusion proteins. J. Virol. 72, 5441–5448 (1998). 9. Gelderblom H.R. Assembly and morphology of HIV: potential effect of structure on viral function. Aids. 5, 617–637 (1991). 10. Mochizuki, H., Schwartz, J.P., Tanaka, K., Brady, R.O. & Reiser, J. High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells. J. Virol. 72, 8873–8883 (1998). 11. Dong, J., Roth, M.G. & Hunter, E. A chimeric avian retrovirus containing the influenza virus hemagglutinin gene has an expanded host range. J. Virol. 66, 7374–7382 (1992). 12. Battini, J.L., Heard, J.M. & Danos, O. Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses. J. Virol. 66, 1468–1475 (1992). 13. Holberg, C.J. et al. Risk factors for respiratory syncytial virus-associated lower respiratory illnesses in the first year of life. Am. J. Epidemiol. 133, 1135–1151 (1991). 14. Johnson, E., Jaax, N., White, J. & Jahrling, P. Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus. Int. J. Exp. Pathol. 76, 227–236 (1995). 15. Snyder, E.Y. et al. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell 68, 33–51 (1992). 16. Yang, Z. et al. Distinct cellular interactions of secreted and transmembrane Ebola virus glycoproteins. Science 279, 1034–1037 (1998). 17. Wool-Lewis, R.J. & Bates, P. Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines. J. Virol. 72,

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