Adenovirus-mediated gene transfer to the ocular surface epithelium.
Gene transfer to the ocular surface epithelium is of potential therapeutic value. It was determined whether a reporter gene can be introduced into the ocular surface epithelium in vitro (human cell lines), ex vivo (human tissues), and in vivo (rats) by treating with a recombinant, replication-deficient, adenovirus type 5. Human and conjunctival cell lines were cultured with various multiplicities of infection (MOI; 3.2x10(-5)-5x10(-1)) of adenovirus vector (Ad5:Adex1CAlacZ) containing the reporter gene lacZ (1.3-2.0x10(4) PFU ml-1). The ex vivo study used human corneal and conjunctival tissues obtained from an eye bank and during surgery. Non-specific upregulation of inflammatory cytokines of conjunctival epithelium infected by Ad5 was assayed and its suppression by steroids. For the in vivo study, Ad5 (5x10(5) PFU, 5-10 microliter) was applied to the eyes of 8-12-week-old cotton rats, which were enucleated 24 and 48 hr later. The maximum lacZ expression in vitro was demonstrated in the corneal epithelial cell line at 7 days (1x10(-1) MOI) and conjunctival epithelial cell line at 2 days (4x10(-4) MOI). Furthermore, lacZ was also expressed in the superficial corneal and conjunctival epithelium in the ex vivo study. IL-6, IL-8, and ICAM-1 expression from conjunctival epithelium by Ad5 was significantly inhibited by treatment with betamethasone (BM). For the in vivo study, only the conjunctival epithelium demonstrated beta-Gal activity at 24 and 48 hr after application. These data indicate that adenovirus vector is capable of directly delivering gene to the corneal and conjunctival epithelium, suggesting a variety of possible gene therapy uses. The concomitant application of steroid eye drops may avoid inflammation.
Tsubota
K
K
Department of Ophthalmology, Tokyo Dental College, Chiba, Japan.
Inoue
H
H
Ando
K
K
Ono
M
M
Yoshino
K
K
Saito
I
I
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001623:Betamethasone / Q000494:pharmacology; D018929:Cell Culture Techniques; D002460:Cell Line; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis; D016209:Interleukin-8 / Q000096:biosynthesis; D007763:Lac Operon; D016205:Sigmodontinae; D015854:Up-Regulation / Q000187:drug effects; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D015850:Interleukin-6; D016209:Interleukin-8; D001623:Betamethasone; D001616:beta-Galactosidase