Somatic Cell and Molecular Genetics, Vol. 17, No. 2, 1991,pp. 185-190

Gene Transfer into Rat Airway Epithelial Cells Using Retroviral Vectors

Christina Stanley, Michael B. Rosenberg, 1 and Theodore Friedmann

Department of Pediatrics and Center for Molecular Genetics, University of California Sc'hool of Medicine, La Jolla, California 92093-0634.

Received 4 December 1990

Abstract--Primary cultures of epithelial cells from adult rat tracheas were maintained in vitro on collagen matrices and were exposed to a murine retrovirus vector e~cpressing the E. coli f3-galactosi- dase gene. Infection was carried out on cells grown as monolayers under medium and on cells' grown on raised platforms. Cells maintained at an air--medium interface were highly susceptible to infection with the vector, showing an efficiency of infection of 20-25%, compared with an efficiency of < 1% for cells grown under medium. Infected f3-galactosidase-expressing cells were seeded into denuded tracheas and were capable of partially repopulating the denuded tracheas grafted subcutaneously into host rats. The susceptibility of these cells' to retroviral infection suggests an approach to the treatment of some pulmonary genetic disorders' such as cystic fibrosis.

INTRODUCTION

The recent cloning of the cystic fibrosis (CF) gene (1) should allow the development of gene therapy approaches to the treatment of this common, severe, and inadequately treated disease. CF is characterized by defective electrolyte transport in exocrine glands and secretory epithelia, and biochem- ical evidence has indicated that the primary defect in CF involves the regulation of apical chloride channels in epithelial cells (2). Although numerous organs are affected, the most serious dysfunction in CF patients is that of the airways, where thick, inspissated mucus leads to life-threatening chronic lung disease. Thus, although it is ultimately important to develop treatments for many of the tissues affected in CF, the most urgent

need is for therapy of the respiratory epithelia.

Two groups have recently demonstrated that transfer of the wild-type CFTR cDNA into immortalized epithelial cell lines derived from CF patients can correct the defect in chloride channel regulation in vitro (3-5). To be effective, gene therapy for CF will require either delivering this cDNA directly to airway epithelia in vivo or transducing primary epithelial cultures in vitro and returning them to the airways. Both ap- proaches require an efficient gene transfer system. Primary cultures of human and rabbit tracheal and bronchial epithelia have been transfected by protoplast fusion (6), strontium phosphate coprecipitation (7), etectroporation (8), and lipofection (9). These methods are all relatively" inefficient,

~Present address: GeneSys Therapeutics Corporation, 11077 North Torrey Pines Road, La Jolla, California 92037. 185

0740-7750/91/0300-018550650/0 © 1991 Plenum Publishing Corporation

186 Rosenberg et al.

with reported stable transfection rates of 10 -3 or less. At present, transducing vectors derived from retroviruses, particularly Molo- ney murine leukemia virus (MoMLV), pro- vide the best understood and most efficient means of expressing foreign genes in cul- tured cells (10, 11). MoMLV-derived vectors have been used to achieve stable foreign gene expression in a variety of cell lines and primary cultures from several species at efficiencies of up to 100%. Although a retroviral vector has been used to transduce human bronchial epithelia that had previ- ously been immortalized by strontium phos- phate transfection with the SV40 large T antigen (12), there have been no reports of the infection of primary cultures of airway epithelia. We demonstrate here that rat tracheal epithelial cells are efficiently in- fected with a retroviraI vector expressing the E. coli [3-galactosidase gene (tacZ), especially when the cells are maintained at an air- medium interface.

MATERIALS AND METHODS

Establishment of Primary Rat Tracheal Epithelial Cultutes. Rat tracheal epithelial cells were isolated from two 200- to 250-g female Sprague-Dawley rats by methods similar to those described previously for hamsters (13). Animals were killed by asphyx- iation with CO2. Their tracheas were re- moved, and the lumens washed with Dulbec- co's phosphate-buffered saline (PBS) containing 50 pog/ml gentamycin and filled with 1% Pronase (Sigma) in Joklik's minimal essential medium containing gentamycin (JMEM). The tracheas were tied closed with dental floss and incubated for 16-20 h at 4°C bathed in JMEM. Cells were flushed out of the tracheas with JMEM, vortexed, and filtered through #500 stainless-steel mesh to remove pieces of undigested tissue. Fetal bovine serum (FBS) was added to a final concentration of 10%. The cells were pel- leted, washed with Ham's F12 medium and

resuspended in a mixture of 1:1 Ham's F12 medium and Dulbecco's modified Eagle medium (DME) supplemented with 1% FBS, 10 txg/ml insulin, 1 pxM hydrocortisone, 2 nM triiodothyronine, 5 ~zg/ml transferrin, 10 ixg/ml endothelial cell growth supplement, 25 ng/ml epidermal growth factor, and 50 p~g/ml gentamycin (complete medium). The cell suspension was plated on collagen gels in ll.3-mm tissue culture wells or on 12-ram raised porous platforms (Millicell-CM, Milli- pore) at a density of 105 cells per well or platform. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. In some cases, the medium above the raised platforms was removed after one day and the cells maintained at the air-liquid interface. The medium in the wells and under the platforms was replaced with fresh medium every two to four days. The collagen gels were prepared by neutralizing 8 ml bovine type 1 collagen (Vitrogen 100, Colla- gen Incorporated, Palo Alto, California) with 1 ml 0.1 N NaOH and 1 ml 10x Dulbecco's PBS. Aliquots of 200 ~xl were applied to each well or platform and potymerized by incuba- tion at 37°C for 2-4 h. The gels then were equilibrated overnight at 37°C with complete medium prior to the addition of cells.

Retrovirus Infections. A retroviral vec- tor, BAG, expressing the E. coli lacZ gene and the Tn5 neomycin phosphotransferase gene (14) was generated from the ~2 eco- tropic producer line (15), as previously de- scribed (16), in DME supplemented with 10% FBS. Virus titer was determined by infecting the Rat1 fibroblast cell line (17) and counting G418-resistant colonies. Tracheal epithelial cells were infected 24 h after plating by ex- posing them from above with virus stock sup- plemented with 4 ~g/ml Polybrene (Sigma) for 2 h at 37°C. Mock-infected control cells were exposed to DME with 10% FBS only. In one experiment, cells were repeatedly treated with virus for up to 16 one-hour exposures. After exposure, the virus stock was removed, the wells or platforms were washed once with

Retrovirus Infection of Airway Epithelial Cells 187

complete medium, medium below the plat- form was replaced with fresh complete medium, and cells were returned to the previous growth conditions. At points rang- ing from 2 to 30 days alter infection, cells were fixed for 15 rain with 1% glutaraldehyde in PBS and stained for [3-galactosidase activity (18). Random fields were photo- graphed and scored for the percentage of stained cells. In some experiments, cells were grown :in complete medium containing 100 ixg/ml G418 beginning 24 h after infection. At this level of G418 selection, uninfected cells died within two days.

Subcutaneous Tracheal Grafts (19). Tra- cheas were dissected from rats, flushed with PBS containing 50 txg/ml gentamycin, frozen at -70°C, thawed at 37°C and flushed again. The freeze-thaw-flush cycle was repeated twice to remove endogenous epithelia. The distal ends of the tracheas were tied off with silk sutures. Cultured epithelial cells were removed from platforms by incubating with 0.1% collagenase in PBS for 1 h at 37°C and then with 0.05% trypsin in PBS containing 1 mg/ml EDTA for 10 rain at 37°C. Cells were harvested by trituration with JMEM, FBS was added to a final concentration of 10%, the cells were pelleted, washed with Ham's F12 medium, and resuspended in complete medium at 106 cells/ml. The tracheal lumens were seeded with approximately 50 ~1 of cell suspensions, and the proximal ends were tied closed below the larynx. The seeded tracheas were grafted subcutaneously into the upper back of host rats.

RESULTS

Rat tracheal epithelial cells were grown on collagen matrices under three conditions: (1) in wells under medium, (2) under medium on a raised porous platform (20, 21), or (3) at an air-medium interface on a raised porous platform (21). After 24 h, the cells were infected from above with a retroviral vector expressing lacZ and neomycin resis-

tance (14) at a multiplicity of infection (MOI) of approximately one virion per cell. Two days after infection, the ceils were stained histochemically for f3-galactosidase activity (1.8). Cells grown at an air-medium interface and therefore maintained only by basal feeding were the most susceptible to infection, showing approximately 20-25% IacZ-positive cells (Fig. tA and Table 1). Cells fed both apically and basally showed a four- to fivefold reduction in infectivity, whereas cells fed only apically showed a further reduction of greater than fivefold in infection efficiency (Table 1). Mock-infected control cells did not stain for [3-galactosidase activity under any growth condition. These differences in infection rates were not due to differences in cellular growth rates, as the cultures were plated at the same densities and reached confluence on the same day. The infection efficiencies of cells on raised platforms were the same whether infection occurred on day 2, 4, or 6 after plating. Increasing the MOI to over 10 by repeated exposure of cells to vector did not change the observed infection rates. Cells infected on day 2 and fed basally have been maintained in culture for up to one month with no significant change in the percentage of cells expressing lacZ. Infected cells could be selected with the neomycin analog G418, resulting in a population in which most cells expressed lacZ (Fig. 1B). All mock-infected cells died within two days of exposure to G418. Cells maintained on raised platforms with basal feeding for one week to one month were stained for [3-galactosidase activity and embedded in paraffin, and 5-/~m transverse sections were prepared. At all times, most of the cells formed a squamous epithelium whether or not they expressed IacZ (Fig. 1C). This is consistent with previous studies indicating that hamster, but not rat, cultured tracheal airway ceils redifferentiate fully into a mucocilliary epithelium under these growth conditions (20, 21).

To determine whether cells grown in

188 Rosenberg et al.

Fig. L Histological detection of [3-galactosidase in airway, epithelial cells, (A) Rat tracheal epithelial ceils maintained at an air-liquid interface and stained for [3-galactosidase 25 days after infection with BAG virus. Cells expressing lacZ are stained blue. (B) Neomycin-resistant cells grown under G418 selection, (C) Transverse section of cells maintained at an air-liquid interface and examined nine days postinfection. The material at the bottom is the platform matrix, and the area between the cells and matrix contains the collagen gel. Cells examined at later time points exhibited a similar morphology. (D) Transverse section of denuded trachea seeded with infected cells grown under G418 selection and examined 14 days after subcutaneous grafting. Bars equal 100 ~xm.

culture with basal feeding and infected with retroviral vectors maintain the capacity for proliferation and mucocilliary differentiation in vivo, infected cells grown with G418 selection were seeded into denuded tra- cheas, which then were grafted subcutane- ously into host rats. Two weeks after grafting, the tracheas were examined histochemically for lacZ activity (Fig. 1D). Although most tracheal regions were not repopulated by epithelial cells, the several repopulated re-

Table 1. Infection of Tracheal Epithlial Cells

[3-Galactosidase-positive cells (%) Feeding

arrangement BAG-infected Mock-infected

Apical < 1 0 Apical and basal 5 0 Basal 20-25 0

gions we observed were lacZ-positive, indicat- ing that the infected cells were capable at least partially of repopulating the airway and that they continued to express the transgene. The cells in these regions, however, had a squamous appearance rather than a pseudo- stratified columnar morphology. Because these experiments used outbred Sprague- Dawtey rats, it is likely that the host immune response was at least partly responsible for the poor survival of grafted cells.

For infection of airway epithelial cells with retrovirus to be feasible in vivo, it would be necessary to deliver large amounts of high-titer virus to a large surface area of the airway. This could be achieved by flooding the airways with a suspension of vector. Alternatively, a patient could inhale aero- solized preparations of virus. To determine

Retrovirus Infection of Airway Epithelial Cells 189

the feasibility of the latter approach, we examined the effects of aerosolization on virus titer. Aerosols were prepared using two nebulizers--a Mountain Medical Equipment Medi-Mist, which uses air pressure for aerosolization, and a DeVilbiss Ultra-Neb 100, which generates aerosols by sonication. The aerosols were collected and allowed to condense in tubes held on ice, and viral titers were measured in the condensates and in the virus stock remaining in the nebulizers (Table 2). The results show that nebulization by sonication reduced virus titer by 98%, whereas nebulization by air pressure caused only a moderate reduction in the virus titer.

DISCUSSION

These studies indicate that cultured rat tracheal epithelial cells can be infected at high efficiency by retroviral vectors and continue to express transgenes for as long as the ceils can be maintained in culture, up to one month. The efficiency of infection is dependent upon the feeding arrangement, with cells grown at an air-liquid interface exhibiting the greatest infection rate. The differences in infectivity suggest that al- though these rat cells do not differentiate into a columnar morphology, they nonethe- less undergo changes in the availability of retroviral receptors or in other membrane characteristics when fed from below. Previ- ous studies have shown that epithelial cells exhibit polarity in their susceptibility to infection by other viruses. For example, infection of the MDCK dog kidney epithelial

TabLe 2. Effect of Aerosolization of Virus Titers

Remaining Condensate in apparatus

Rel. Rel. Nebulizer Titer titer ~ Titer titer ~

Medi-Mist 2.9 x 104 0.32 5.3 × 104 0.58 Ultra-Neb100 2.0 x 10 a 0.02 5.5 × 104 0.60

"Titer relative to control vector stock (9.1 x 104/ml).

line by SV40 occurs through the apical membrane only (22), whereas infection of these cells by canine parvovirus has a basolateral restriction (23), reflecting polar- ized distributions of the viral receptors. Further studies are necessary to determine whether the ecotropic retroviral receptor also exhibits a polarized distribution. It is also possible that the different infection rates reflect differences in epithelial subpopula- tions obtained under the three growth conditions.

Because airway epitheliaI cells can be efficiently infected by retroviral vectors and because retrovirus stocks can be aerosolized with only a moderate reduction in titer, delivery of an aerosolized vector expressing the CF transmembrane conductance regula- tor (24) may become a rational approach to gene therapy for CF. Since the human airway epithelium has an extremely large surface area, it would be necessary to generate vector stocks with very high titers. The recent development of the "ping pong" method for increasing vector titer by cocuttivating ampho- tropic and ecotropic producer cells (25) may prove valuable tbr this purpose. Another important consideration is that airway epithe- lial cells normally exhibit low mitotic activity. Ciliated cells are a terminally differentiated, postmitotic population, and although mu- cous and basal cells are known to be mitotically active, the labeling indices of these cells in adult mice with [3H]thymidine are only 0.8% and 0.4%, respectively (26). However, the lung can be classified as a conditional renewal system (26). Ciliated cells are extremely sensitive to injury caused by- irritation or infection (27), and mucous cell proliferation is the major response after injury (26). Thus, the airways of CF patients have greater mitotic activities than those of unaffected people. In addition, lung develop- ment in humans continues for about eight years after birth, with linear growth observed during this period (28). Therefore, it is possible that under the appropriate condi-

190 Rosenberg et al.

tions, the airways may present a suitable target cell population for retrovirus infec- tion.

LITERATURE CITED

1. Rommens, J.M., Iannuzzi, M.C., Kerem, B.-S., Drumm, M.L., Melmer, G., Dean, M., Rozmahel, R., Cole, J.L., Kennedy, D., Hidaka, N., Zsiga, M., Buchwald, M., Riordan, J,R., Tsui, L.-C, and Collins, F.S. (1989). Science 245:1059-1065.

2. McPherson, M.A., and Dormer, R.L. (1987). Biosci. Rep. 7:167-185.

3. Drumm, M.L., Pope, H.A., Cliff, W.H., Rommens, J.M., Marvin, S.A., Tsui, L.-C., Collins, F.S, Frizzell, R.A., and Wilson, J.M. (t990). Ceil 62:1227-1233.

4. Gregory, R.J., Cheng, S.H., Rich, D.P., Marshall, J., Paul, S., Hehir, K., Ostedgaard, L., Klinger, K.W., Welsh, M.J., and Smith, A.E. (1990). Nature 347:382-386.

5. Rich, D.P., Anderson, M.P., Gregory, RJ., Cheng, S.H., Paul, S., Jefferson, D.M., McCann, J.D., Klinger, K.W., Smith, A.E, and Welsh, MJ. (1990). Nature 347:358-363.

6. Yoakum, G.H., Lechner, J.F., Gabrielson, E.W., Korba, B.E., Malan-Shibley, L., Wiley, J.C., Vale- rio, M.G., Shamsuddin, A.M., Trump, B.F., and Harris, C.C. (1985). Science 227:1174-1179.

7. Brash, D.E., Reddel, R.R., Quanrud, M., -fang, K., Farrell, M.P., and Harris, C.C. (1987). MoL Ceil. Biol. 7:2031-2034.

8. Iannuzzi, M.C., Weber, J.L., Yankaskas, J, Boucher, R., and Collins, F.S. (1988). Am. Rev. Respir. Dis. 138:965-968.

9. Luo, L., Zeitlin, P.L., Guggino, W.B., and Craig, R.W. (1989). PflugersArch. 415:198-203.

10. Temin, H.M. (1986). In Gene Transfel; (ed.) Kucherlapati, R. (Plenum Press, New York), pp. 149-187.

11. Eglitis, M.A., and Anderson, W.F. (1988). BioTech- niques 6:608-614.

12. Amstad, P., Reddel, R.R., Pfeifer, A., Malan-

Shibley, L., Mark, G.E, III, and Harris, C.C. (1988). MoL Carcinoger, 1:151-160.

13. Lee, T.-C., Wu, R., Brody, A.R, Barrett, J.C., and Nettesheim, P. (1984). Exp. Lung Res. 6:27-45.

14. Price, J., Turner, D., and Cepko, C. (1987). Proc. Natl. Acad. Sci. U.S.A. 84:156-160.

15. Mann, R., Mulligan, R.C., and Baltimore, D. (1983). Cell 33:153-159.

16. Shimohama, S, Rosenberg, M.B., Fagan, A.M., Wolff, J.A, Short, M.P., Breakefield, X.O., Fried- mann, T., and Gage, F.H. (1989). Mol. Brain Res. 5:271-278.

17. Mishra, N.K., and Ryan, W.L. (1973), lnt. J. Cancer 11:123-130.

18. Turner, D.L., and Cepko, C.L. (1987). Nature 328:131-136.

19. Brody, A.R., Hook, G.E.R., Cameron, G.S., Jetten, A.M., Butterick, C.J., and Nettesheim, P. (1987). Lab. Invest. 57:219-229.

20. Moiler, P.C., Partridge, L.R., Cox, R., Pellegrini, V., and Ritchie, D.G. (1987). Tissue Cell 19:783- 791.

21. Whitcutt, M.J., Adler, K.B., and Wu, R. (1988). In Vitro Cell. Dev. Biol. 24:420M28.

22. Clayson, E.T., and Compans, R.W. (1988). MoL Cell BioL 8:3391-3396.

23. Basak, S, and Compans, R.W. (1989). J. Virol. 63:3164-3167.

24. Riordan, J.R., Rommens, J.M,, Kerem, B.-S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M.L., Iannuzzi, M.C., Collins, F.S., and Tsui, L.-C. (1989). Science 245:1066-1073.

25. Bodine, D.M., McDonagh, K.T., Brandt, SJ., Ney, P.A., Agricola, B., Byrne, E., and Nienhuis, A.W. (1990). Proc. Natl. Acad. Sci. U.S.A. 87:3738-3742.

26. Evans, MJ., and Shami, S.G. (1989). In Lung Cell Biology, (ed.) Massaro, D. (Marcel Dekker, New York), pp. 1-36.

27. Sturgess, J.M. (1989). In Lung Cell Biology, (ed.) Massaro, D. (Marcel Dekker, New York), pp. 115-151.

28. Farrell, P.M. (1982). In Lung Development: Biologi- cal and Clinical Perspectives, Vol. I, (ed.) Farrell, P.M. (Academic Press, New York), pp. 13-25.