New strategy for in vivo transgene expression in corneal epithelial progenitor cells.
OBJECTIVE
Efficient in vivo gene transfer into corneal epithelial cells and stable transgene expression would have broad clinical applications. We therefore assessed the capacity of adenoviral, adeno-associated viral (AAV) and lentiviral vectors encoding enhanced green fluorescent protein (EGFP) to transduce rat corneal epithelial progenitor cells.


METHODS
Superficial cells of the corneal epithelium were shaved, after which 20 microl of the respective vector solutions were inoculated onto the basal cell layer of the limbal epithelium for 30 min. Results. Three days after transduction, fluorescence microscopic examination revealed the presence of EGFP+ cells in all corneas, irrespective of the vector used; however, EGFP+ cells were undetectable in corneas transduced with adenoviral and AAV vectors after 2 and 4 weeks, respectively. By contrast, EGFP+ cells were still detected among cells transduced with lentiviral vector 6 weeks after transduction. DAPI (4', 6-diamidino-2-phenylindole) staining confirmed that it was the basal layer cells that continued to express EGFP throughout the 6-week period.


CONCLUSIONS
The use of a lentiviral vector for in vivo transfer of foreign genes into corneal epithelial stem and transient amplifying cells may represent a new and effective approach to the treatment of corneal disease, as well as to the study of the biology of these stem cells.
Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan. tutomu@nms.ac.jp
Tsutomu
T
Igarashi
Koichi
K
Miyake
Noriko
N
Suzuki
Ko
K
Kato
Hiroshi
H
Takahashi
Kunitoshi
K
Ohara
Takashi
T
Shimada
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D051381:Rats; D011916:Rats, Inbred F344; D013234:Stem Cells / Q000378:metabolism*; D019076:Transgenes
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins